Materials and methods for respiratory disease control in canines

ABSTRACT

The subject invention pertains to isolated influenza virus that is capable of infecting canids and causing respiratory disease in the canid. The subject invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the present invention. The subject invention also pertains to compositions and methods for identifying a virus of the invention and diagnosing infection of an animal with a virus of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 13/066,651, filed Apr.19, 2011, which is a divisional of U.S. Ser. No. 11/584,818, filed Oct.19, 2006, now U.S. Pat. No. 7,959,929, which is a continuation-in-partof U.S. Ser. No. 11/409,416, filed Apr. 21, 2006, now abandoned, whichclaims priority from U.S. Ser. No. 60/673,443, filed Apr. 21, 2005; andU.S. Ser. No. 11/584,818 claims priority to U.S. Ser. Nos. 60/728,449,filed Oct. 19, 2005; 60/754,881, filed Dec. 29, 2005; 60/759,162, filedJan. 14, 2006; 60/761,451, filed Jan. 23, 2006; and 60/779,080, filedMar. 3, 2006, the disclosure of each of which is hereby incorporated byreference herein in its entirety, including any brief summary, detaileddescriptions of the invention, examples, claims, abstract, figures,tables, nucleic acid sequences, amino acid sequences, and drawings.

The Sequence Listing for this application is labeled “2DD9068.TXT” whichwas created on Oct. 3, 2014 and is 400 KB. The entire contents of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

“Kennel cough” or infectious tracheobronchitis (ITB) is an acute,contagious respiratory infection in dogs characterized mainly bycoughing (Ford et al, 1998). Canine ITB is considered one of the mostprevalent infectious respiratory diseases of dogs worldwide, andoutbreaks can reach epidemic proportions when dogs are housed inhigh-density population environments such as kennels. Most outbreaks aredue to direct dog-to-dog contact or aerosolization of respiratorysecretions (Ford et al, 1998). The clinical signs are caused byinfection with one or a combination of bacterial and viral agents thatcolonize the epithelium of the upper and lower respiratory tract. Canineparainfluenza virus (CPiV) and Bordetella bronchiseptica bacteria arethe most common organisms isolated from affected dogs, but several otherviruses such as canine distemper virus (CDV) and canine adenoviruses-1and -2 (CAV-1, CAV-2), along with bacteria such as Streptococcus sp.,Pasteurella multicoda and Escherichia coli, can influence the clinicalcourse and outcome (Ford et al, 1998). While outbreaks occur mostefficiently and rapidly in high-density populations with high morbidity,complicated respiratory infections and death are uncommon. Althoughlife-threatening secondary bacterial pneumonia can develop, the majorityof ITB cases are self-limiting and resolve without any treatment (Fordet al, 1998).

In July 1992, a respiratory infection presumed to be “kennel cough”became epidemic at several greyhound tracks in New England, Florida,West Virginia, Wisconsin, Kansas, Colorado, Oklahoma and Arizona.According to veterinarians, most of the affected dogs had a mild coughthat resolved, but more than a dozen greyhounds developed an acutehemorrhagic pneumonia followed by rapid death (Greyhound Daily News,1999).

In late 1998 to early 1999, several outbreaks of “kennel cough” occurredin racing greyhound kennels across the country, resulting in mandatoryclosure of tracks and quarantine of all racing greyhounds in the U.S.for several weeks (Greyhound Daily News, 1999). At one track in Florida(Palm Beach Kennel Club), coughing was recorded in nearly 40% of the dogpopulation on a single day (Personal communication from Dr. WilliamDuggar). Similar to the outbreak in 1992, the coughing resolved in mostgreyhounds, but 10 dogs in Florida died from a hemorrhagic pneumoniasyndrome uncharacteristic of “kennel cough” (Putnam, 1999).

In March-April 2003, another outbreak of “kennel cough” occurred atgreyhound tracks in the eastern U.S. The outbreak is believed to haveoriginated in kennels at four tracks in Florida and caused thesuspension of racing and quarantine of dogs for almost three weeks.Nearly 25% of the dogs at the track in West Palm Beach were affected,while almost 50% of the 1400 dogs at Derby Lane in St. Petersburgdeveloped coughing. Again, most dogs recovered, but several dogs havedied from the respiratory infection. The estimated economic impact ofthe respiratory outbreak at the Derby Lane track alone was $2 million.

There are no published reports documenting the etiology orclinicopathology of the “kennel cough” epidemics in racing greyhoundkennels in 1992, 1998-1999, or 2003. The assumption has been that theinfections were due to CPiV and/or B. bronchiseptica, the two mostcommon causes of kennel cough. Unsubstantiated communications such asweb sites have attributed the fatal hemorrhagic pneumonias reported insome of the coughing dogs to infection with β-hemolytic Streptococcusequi subspecies zooepidemicus, and refer to the syndrome as “caninestreptococcal toxic shock.”

Transmission of virus from one host species to another is a crucialfeature of the ecology and epidemiology of influenza virus (Webster,1998). Two basic mechanisms of interspecies transmission of influenzavirus are possible (Webster et al., 1992; Lipatov et al., 2004). One isthe direct transfer of an essentially unaltered virus from one speciesto another. Examples of this mechanism include the recent humaninfections with the H5N1 subtype of avian influenza virus (Subbarao etal., 1998; Peiris et al., 2004; Guan et al., 2004) and possibly thepandemic of 1918, known as Spanish flu (Reid et al., 2004). The secondmechanism is a consequence of the segmented nature of the influenzagenome. Co-infection of a host with viruses from different species canresult in reassortment of the segmented viral genes and the generationof a recombinant with the ability to infect other species. For example,novel viruses generated by gene reassortment between avian and humaninfluenza viruses resulted in human influenza pandemics in 1957 and 1968(Webster et al., 1992; Lipatov et al., 2004; Kawaoka et al., 1989).

Most direct transmissions of unaltered influenza viruses from thenatural host species to a different species are terminal events becausesustained transmission between individuals of the new species fails tooccur. Multiple virus-host interactions are necessary for replicationand horizontal transmission and provide a formidable barrier toperpetuation of influenza viruses in the new host (Webby et al., 2004).Therefore, establishment of new host-specific lineages of influenzavirus is uncommon and has only occurred in domestic poultry, pigs,horses, and humans (Webster et al., 1992; Lipatov et al., 2004).

Because of the serious nature of influenza virus infection, thereremains a need for methods for diagnosing, preventing, and treatinginfection by influenza virus.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to isolated influenza virus that iscapable of infecting canids and causing respiratory disease in thecanid. The subject invention also pertains to compositions and methodsfor inducing an immune response against an influenza virus of thepresent invention. The subject invention also pertains to compositionsand methods for identifying a virus of the invention and diagnosinginfection of an animal with a virus of the invention.

One aspect of the invention relates to vaccines and methods forprotecting canines from canine influenza, kits comprising such vaccines,and methods for using such vaccines. This protection includespreventing, reducing the risk of, delaying the onset of, reducing thespread of, ameliorating, suppressing, and/or eradicating the influenzaand/or one or more (typically two or more) of its symptoms. It isbelieved that the vaccines, kits, and methods of this invention aregenerally suitable for use with canines. Canines include wild, zoo, anddomestic canines, such as wolves, coyotes, and foxes. Canines alsoinclude dogs, particularly domestic dogs, such as, for example,pure-bred and/or mongrel companion dogs, show dogs, working dogs,herding dogs, hunting dogs, guard dogs, police dogs, racing dogs, and/orlaboratory dogs.

This invention is also directed, in part, to a method for protecting acanine from an influenza virus infection (i.e., preventing, reducing therisk of, delaying the onset of, suppressing, ameliorating, oreradicating an influenza virus infection). The method comprisesadministering a therapeutically effective amount of a vaccine thatcomprises at least one equine influenza virus antigen, at least one H3influenza virus antigen, and/or at least one H7 influenza virus antigen.

This invention also is directed, in part, to a method for protecting acanine from respiratory lesions (i.e., preventing, reducing the risk of,delaying the onset of, suppressing, ameliorating, or eradicatingrespiratory lesions) caused by canine influenza virus. The methodcomprises administering to the canine a therapeutically effective amountof a vaccine that comprises at least one equine influenza virus antigen,at least one H3 influenza virus antigen, and/or at least one H7influenza virus antigen.

This invention also is directed, in part, to a method for protecting acanine from having canine influenza virus in nasal or oral secretion(i.e., preventing, reducing the risk of, delaying the onset of,suppressing, ameliorating, or eradicating canine influenza virus innasal or oral secretion) caused by canine influenza virus infection. Themethod comprises administering to the canine a therapeutically effectiveamount of a vaccine that comprises at least one equine influenza virusantigen, at least one H3 influenza virus antigen, and/or at least one H7influenza virus antigen.

This invention also is directed, in part, to a canine influenza vaccine.In some embodiments, for example, the vaccine comprises atherapeutically effective amount of at least one equine influenza virusantigen, at least one H3 influenza virus antigen, and/or at least one H7influenza virus antigen.

This invention also is directed, in part, to a kit for protecting acanine from influenza virus infection. The kit comprises atherapeutically effective amount of a vaccine that comprises at leastone equine influenza virus antigen, at least one H3 influenza virusantigen, and/or at least one H7 influenza virus antigen. In addition,the kit comprises at least one of the following:

-   -   an apparatus for administering the vaccine to the canine,    -   a pharmaceutically acceptable excipient that aids in        administering the vaccine to the canine,    -   a pharmaceutically acceptable excipient that enhances the        canine's immune response to the vaccine,    -   a food to be consumed by the canine simultaneously with the        vaccine, and/or    -   a treat to be consumed by the canine simultaneously with the        vaccine.

Further benefits of Applicants' invention will be apparent to oneskilled in the art from reading this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Patent Office upon request andpayment of the necessary fee.

FIGS. 1A-1B show phylogenetic relationships among the hemagglutiningenes. FIG. 1A shows a tree of HA genes from representative canine,human, avian, swine, and equine isolates, includingA/Budgerigar/Hokkaido/1/77 (H4) as outgroup. FIG. 1B shows a tree of thecanine influenza virus HA genes with contemporary and older equine HAgenes, using A/Duck/Ukraine/63 (H3) as outgroup. Phylogenetic trees wereinferred from nucleotide sequences by the neighbor joining method andbootstrap analysis values 90% are shown. The bar denotes the number ofnucleotide changes per unit length of the horizontal tree branches.

FIGS. 2A-2B show immunohistochemical detection of influenza H3 antigenin the lungs. Lung tissue sections were probed with a mouse monoclonalantibody to H3 hemagglutinin and binding was detected byimmunoperoxidase reaction (brown precipitate). FIG. 2A shows bronchialepithelium from a greyhound with spontaneous disease. Viral H3 antigenwas detected in bronchial epithelial cell cytoplasm and in macrophagesin airway lumens and in alveolar spaces. FIG. 2B shows bronchialepithelium from a dog 5 days after inoculation withA/canine/Florida/43/2004 (H3N8). Viral H3 antigen was detected inbronchial epithelial cell cytoplasm. Scale bar, 66 μm.

FIGS. 3A and 3B show the characteristic histological changes in thebronchi of greyhounds that died from hemorrhagic pneumonia associatedwith influenza virus infection. The tissues are stained with H&E. FIG.3A: Normal bronchus with ciliated epithelial cells, mucous cells, andbasal cells. FIG. 3B: Bronchus from a greyhound with spontaneousinfluenza. There is necrosis and erosion of the bronchial ciliatedepithelial cells. Scale bar, 100 μm.

FIGS. 4A-4B shows phylogenetic relationships among the H3 hemagglutiningenes. FIG. 4A shows a phylogenetic tree of the canine influenza virusHA genes with contemporary and older equine HA genes. FIG. 4B shows aphylogenetic tree of the canine influenza virus HA protein withcontemporary and older equine HA. Phylogenetic trees were inferred fromgenetic or amino acid sequences by the neighbor joining method andbootstrap analysis values ≧80% are shown. The bar denotes the number ofamino acid changes per unit length of the horizontal tree branches.

FIG. 5 shows Influenza virus H3 protein in epithelial cells of bronchiand bronchial glands in lungs of dogs that died of pneumonia associatedwith influenza virus infection. Upper panels: Erosion of ciliatedbronchial epithelial cells in bronchi. Tissues were stained with H&E.Lower panels: Influenza virus H3 protein in the cytoplasm of bronchial(left) and bronchial gland (right) epithelial cells. Tissues werestained with a monoclonal antibody to influenza H3 detected byimmunoperoxidase reaction (brown precipitate) and counterstained withhematoxylin.

FIGS. 6A-6D show amplification plots of H3 and Matrix genes (FIG. 6A andFIG. 6B) obtained from the amplification of 10-fold serially diluted invitro transcribed RNA standards. Standard curves of H3 and Matrix genes(FIG. 6C and FIG. 6D) constructed by plotting the log of starting RNAconcentrations against the threshold cycle (Ct) obtained from eachdilution.

FIG. 7 shows sensitivity of Directigen Flu A was tested with 10-foldserially diluted virus stocks including A/Wyoming/3/2003 andA/canine/FL/242/2003. The purple triangle indicates positive result.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding a PB2 protein that can be used according to thepresent invention.

SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.

SEQ ID NO: 3 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding a PB1 protein that can be used according to thepresent invention.

SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.

SEQ ID NO: 5 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding a PA protein that can be used according to thepresent invention.

SEQ ID NO: 6 is the amino acid sequence encoded by SEQ ID NO: 5.

SEQ ID NO: 7 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding an NS protein that can be used according to thepresent invention.

SEQ ID NO: 8 is the amino acid sequence encoded by SEQ ID NO: 7.

SEQ ID NO: 9 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding an NP protein that can be used according to thepresent invention.

SEQ ID NO: 10 is the amino acid sequence encoded by SEQ ID NO: 9.

SEQ ID NO: 11 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding an NA protein that can be used according to thepresent invention.

SEQ ID NO: 12 is the amino acid sequence encoded by SEQ ID NO: 11.

SEQ ID NO: 13 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding an MA protein that can be used according to thepresent invention.

SEQ ID NO: 14 is the amino acid sequence encoded by SEQ ID NO: 13.

SEQ ID NO: 15 is a nucleotide sequence of a canine influenza virus(Florida/43/04) encoding an HA protein that can be used according to thepresent invention.

SEQ ID NO: 16 is the amino acid sequence encoded by SEQ ID NO: 15.

SEQ ID NO: 17 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding a PB2 protein that can be used according to thepresent invention.

SEQ ID NO: 18 is the amino acid sequence encoded by SEQ ID NO: 17.

SEQ ID NO: 19 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding a PB1 protein that can be used according to thepresent invention.

SEQ ID NO: 20 is the amino acid sequence encoded by SEQ ID NO: 19.

SEQ ID NO: 21 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding a PA protein that can be used according to thepresent invention.

SEQ ID NO: 22 is the amino acid sequence encoded by SEQ ID NO: 21.

SEQ ID NO: 23 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding an NS protein that can be used according to thepresent invention.

SEQ ID NO: 24 is the amino acid sequence encoded by SEQ ID NO: 23.

SEQ ID NO: 25 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding an NP protein that can be used according to thepresent invention.

SEQ ID NO: 26 is the amino acid sequence encoded by SEQ ID NO: 25.

SEQ ID NO: 27 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding an NA protein that can be used according to thepresent invention.

SEQ ID NO: 28 is the amino acid sequence encoded by SEQ ID NO: 27.

SEQ ID NO: 29 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding an MA protein that can be used according to thepresent invention.

SEQ ID NO: 30 is the amino acid sequence encoded by SEQ ID NO: 29.

SEQ ID NO: 31 is a nucleotide sequence of a canine influenza virus(FL/242/03) encoding an HA protein that can be used according to thepresent invention.

SEQ ID NO: 32 is the amino acid sequence encoded by SEQ ID NO: 31.

SEQ ID NO: 33 is the mature form of the HA protein shown in SEQ ID NO:16 wherein the N-terminal 16 amino acid signal sequence has beenremoved.

SEQ ID NO: 34 is the mature form of the HA protein shown in SEQ ID NO:32 wherein the N-terminal 16 amino acid signal sequence has beenremoved.

SEQ ID NO: 35 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 36 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 37 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 38 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 39 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 41 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 42 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 43 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 44 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 45 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 46 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 47 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding a PB2 protein that can be used according to thepresent invention.

SEQ ID NO: 48 is the amino acid sequence encoded by SEQ ID NO: 47.

SEQ ID NO: 49 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding a PB1 protein that can be used according to thepresent invention.

SEQ ID NO: 50 is the amino acid sequence encoded by SEQ ID NO: 49.

SEQ ID NO: 51 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding a PA protein that can be used according to thepresent invention.

SEQ ID NO: 52 is the amino acid sequence encoded by SEQ ID NO: 51.

SEQ ID NO: 53 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding an NS protein that can be used according to thepresent invention.

SEQ ID NO: 54 is the amino acid sequence encoded by SEQ ID NO: 53.

SEQ ID NO: 55 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding an NP protein that can be used according to thepresent invention.

SEQ ID NO: 56 is the amino acid sequence encoded by SEQ ID NO: 55.

SEQ ID NO: 57 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding an NA protein that can be used according to thepresent invention.

SEQ ID NO: 58 is the amino acid sequence encoded by SEQ ID NO: 57.

SEQ ID NO: 59 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding an MA protein that can be used according to thepresent invention.

SEQ ID NO: 60 is the amino acid sequence encoded by SEQ ID NO: 59.

SEQ ID NO: 61 is a nucleotide sequence of a canine influenza virus(Miami/2005) encoding an HA protein that can be used according to thepresent invention.

SEQ ID NO: 62 is the amino acid sequence encoded by SEQ ID NO: 61.

SEQ ID NO: 63 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding a PB2 protein that can be used according tothe present invention.

SEQ ID NO: 64 is the amino acid sequence encoded by SEQ ID NO: 63.

SEQ ID NO: 65 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding a PB1 protein that can be used according tothe present invention.

SEQ ID NO: 66 is the amino acid sequence encoded by SEQ ID NO: 65.

SEQ ID NO: 67 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding a PA protein that can be used according tothe present invention.

SEQ ID NO: 68 is the amino acid sequence encoded by SEQ ID NO: 67.

SEQ ID NO: 69 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding an NS protein that can be used according tothe present invention.

SEQ ID NO: 70 is the amino acid sequence encoded by SEQ ID NO: 69.

SEQ ID NO: 71 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding an NP protein that can be used according tothe present invention.

SEQ ID NO: 72 is the amino acid sequence encoded by SEQ ID NO: 71.

SEQ ID NO: 73 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding an NA protein that can be used according tothe present invention.

SEQ ID NO: 74 is the amino acid sequence encoded by SEQ ID NO: 73.

SEQ ID NO: 75 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding an MA protein that can be used according tothe present invention.

SEQ ID NO: 76 is the amino acid sequence encoded by SEQ ID NO: 75.

SEQ ID NO: 77 is a nucleotide sequence of a canine influenza virus(Jacksonville/2005) encoding an HA protein that can be used according tothe present invention.

SEQ ID NO: 78 is the amino acid sequence encoded by SEQ ID NO: 77.

SEQ ID NO: 79 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 80 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 81 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 82 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 83 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 84 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 85 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 86 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 87 is an oligonucleotide that can be used according to thepresent invention.

SEQ ID NO: 88 is an oligonucleotide that can be used according to thepresent invention.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns isolated influenza virus that is capableof infecting canids and causing respiratory disease. In one embodiment,an influenza virus of the invention comprises a polynucleotide whichencodes a protein having an amino acid sequence shown in any of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, or afunctional and/or immunogenic fragment or variant thereof. In a specificembodiment, the polynucleotide comprises the nucleotide sequence shownin any of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or77, or a fragment or variant thereof. Influenza virus of the presentinvention can have an HA subtype of H1, H2, H3, H4, H5, H6, H7, H8, andH9, H10, H11, H12, H13, H14, H15, or H16 or an NA subtype of N1, N2, N3,N4, N5, N6, N7, N8, OR N9. In a specific embodiment, an influenza virusof the present invention is a subtype H3. Virus can be isolated frominfected dogs and cultured in cells or eggs according to methodsdescribed herein. In an exemplified embodiment, the influenza virus isan influenza A virus.

The subject invention also concerns polynucleotides that comprise all orpart of a gene or genes or a genomic segment of an influenza virus ofthe present invention. In one embodiment, a polynucleotide of theinvention comprises an influenza hemagglutinin (HA) gene, neuraminidase(NA) gene, nucleoprotein (NP) gene, matrix protein (MA or M) gene,polymerase basic (PB) protein gene, polymerase acidic (PA) protein gene,non-structural (NS) protein gene, or a functional fragment or variant ofany of these genes. In a specific embodiment, a polynucleotide of theinvention comprises the hemagglutinin (HA) gene, or a functionalfragment or variant thereof. In a further embodiment, the HA geneencodes a hemagglutinin protein having one or more of the following: aserine at position 83; a leucine at position 222; a threonine atposition 328; and/or a threonine at position 483, versus the amino acidsequence of equine H3 consensus sequence. In one embodiment, the HA geneencodes a polypeptide having an amino acid sequence shown in SEQ ID NOs:16, 32, 62, or 78, or a functional and/or immunogenic fragment orvariant thereof. In a specific embodiment, the HA gene comprises anucleotide sequence shown in SEQ ID NOs: 15, 31, 61, or 77.

In one embodiment, a polynucleotide of the invention encodes apolypeptide having the amino acid sequence shown in any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, or afunctional and/or immunogenic fragment or variant thereof. In a specificembodiment, the polynucleotide encoding the amino acid sequence shown inSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or78, comprises the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, respectively, or a sequenceencoding a functional and/or immunogenic fragment or variant of any ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or78. Thus, the subject invention concerns polynucleotide sequencescomprising the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, or a fragment or variant,including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, or 77. In a further specific embodiment, apolynucleotide of the invention can comprise: Nucleotides 1-2271 of SEQID NO: 3; Nucleotides 1-2148 of SEQ ID NO: 5; Nucleotides 1-657 of SEQID NO: 7; Nucleotides 1-1494 of SEQ ID NO: 9; Nucleotides 1-1410 of SEQID NO: 11; Nucleotides 1-756 of SEQ ID NO: 13; Nucleotides 1-1695 of SEQID NO: 15; Nucleotides 1-2271 of SEQ ID NO: 19; Nucleotides 1-2148 ofSEQ ID NO: 21; Nucleotides 1-657 of SEQ ID NO: 23; Nucleotides 1-1494 ofSEQ ID NO: 25; Nucleotides 1-756 of SEQ ID NO: 29; Nucleotides 1-1695 ofSEQ ID NO: 31; Nucleotides 1-2277 of SEQ ID NO: 47; Nucleotides 1-2271of SEQ ID NO: 49; Nucleotides 1-2148 of SEQ ID NO: 51; Nucleotides 1-690of SEQ ID NO: 53; Nucleotides 1-1494 of SEQ ID NO: 55; Nucleotides1-1410 of SEQ ID NO: 57; Nucleotides 1-756 of SEQ ID NO: 59; Nucleotides1-1695 of SEQ ID NO: 61; Nucleotides 1-2277 of SEQ ID NO: 63;Nucleotides 1-2271 of SEQ ID NO: 65; Nucleotides 1-2148 of SEQ ID NO:67; Nucleotides 1-690 of SEQ ID NO: 69; Nucleotides 1-1494 of SEQ ID NO:71; Nucleotides 1-1410 of SEQ ID NO: 73; Nucleotides 1-756 of SEQ ID NO:75; and Nucleotides 1-1695 of SEQ ID NO: 77. Nucleotide and amino acidsequences of viral polynucleotide and polypeptide sequences contemplatedwithin the scope of the present invention have also been deposited withGenBank at accession Nos. DQ124147 through DQ124161 and DQ124190, thedisclosure of which is incorporated herein by reference.

The subject invention also concerns polypeptides encoded bypolynucleotides of an influenza virus of the present invention. Thesubject invention also concerns functional and/or immunogenic fragmentsand variants of the subject polypeptides. Polypeptides contemplatedinclude HA protein, NA protein, NS protein, nucleoprotein, polymerasebasic protein, polymerase acidic protein, and matrix protein of aninfluenza virus of the invention. In an exemplified embodiment, apolypeptide of the invention has an amino acid sequence shown in any ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or78, or a functional and/or immunogenic fragment or variant thereof.

The subject invention also concerns polynucleotide expression constructscomprising a polynucleotide sequence of the present invention. In oneembodiment, an expression construct of the invention comprises apolynucleotide sequence encoding a polypeptide comprising an amino acidsequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, or 78, or a functional and/or immunogenic fragmentor variant thereof. In a specific embodiment, the polynucleotideencoding the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78 comprises the nucleotidesequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, or 77, respectively, or a sequence encoding a functional and/orimmunogenic fragment or variant of any of SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78. Thus, the subjectinvention concerns expression constructs comprising a polynucleotidesequence comprising the nucleotide sequence shown in any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, or a fragment orvariant, including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77. In a preferredembodiment, an expression construct of the present invention providesfor overexpression of an operably linked polynucleotide of theinvention.

Expression constructs of the invention generally include regulatoryelements that are functional in the intended host cell in which theexpression construct is to be expressed. Thus, a person of ordinaryskill in the art can select regulatory elements for use in, for example,human host cells, mammalian host cells, insect host cells, yeast hostcells, bacterial host cells, and plant host cells. In one embodiment,the regulatory elements are ones that are functional in canine cells.Regulatory elements include promoters, transcription terminationsequences, translation termination sequences, enhancers, andpolyadenylation elements. As used herein, the term “expressionconstruct” refers to a combination of nucleic acid sequences thatprovides for transcription of an operably linked nucleic acid sequence.As used herein, the term “operably linked” refers to a juxtaposition ofthe components described wherein the components are in a relationshipthat permits them to function in their intended manner. In general,operably linked components are in contiguous relation.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding apolypeptide of the invention. Promoters can be incorporated into apolynucleotide using standard techniques known in the art. Multiplecopies of promoters or multiple promoters can be used in an expressionconstruct of the invention. In a preferred embodiment, a promoter can bepositioned about the same distance from the transcription start site inthe expression construct as it is from the transcription start site inits natural genetic environment. Some variation in this distance ispermitted without substantial decrease in promoter activity. Atranscription start site is typically included in the expressionconstruct. Preferably, the promoter associated with an expressionconstruct of the invention provides for overexpression of an operablylinked polynucleotide of the invention.

Promoters for use with an expression construct of the invention ineukaryotic cells can be of viral or cellular origin. Viral promotersinclude, but are not limited to, cytomegalovirus (CMV) gene promoters,SV40 early or late promoters, or Rous sarcoma virus (RSV) genepromoters. Promoters of cellular origin include, but are not limited to,desmin gene promoter and actin gene promoter Promoters suitable for usewith an expression construct of the invention in yeast cells include,but are not limited to, 3-phosphoglycerate kinase promoter,glyceraldehyde-3-phosphate dehydrogenase promoter, metallothioneinpromoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

If the expression construct is to be provided in or introduced into aplant cell, then plant viral promoters, such as, for example, acauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35Spromoter (see, for example U.S. Pat. No. 5,106,739 and An, 1997)) or aCaMV 19S promoter can be used. Other promoters that can be used forexpression constructs in plants include, for example, proliferapromoter, Ap3 promoter, heat shock promoters, T-DNA 1′- or 2′-promoterof A. tumefaciens, polygalacturonase promoter, chalcone synthase A(CHS-A) promoter from petunia, tobacco PR-1a promoter, ubiquitinpromoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al.,1993), maize WipI promoter, maize trpA gene promoter (U.S. Pat. No.5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S.Pat. No. 5,034,322) can also be used. Root-specific promoters, such asany of the promoter sequences described in U.S. Pat. No. 6,455,760 orU.S. Pat. No. 6,696,623, or in published U.S. patent application Nos.20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or20040123349, can be used with an expression construct of the invention.Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOSpromoter), developmentally-regulated promoters, and inducible promoters(such as those promoters than can be induced by heat, light, hormones,or chemicals) are also contemplated for use with polynucleotideexpression constructs of the invention. Tissue-specific promoters, forexample fruit-specific promoters, such as the E8 promoter of tomato(accession number: AF515784; Good et al. (1994)) can also be used.Seed-specific promoters such as the promoter from a β-phaseolin gene(for example, of kidney bean) or a glycinin gene (for example, ofsoybean), and others, can also be used.

For expression in prokaryotic systems, an expression construct of theinvention can comprise promoters such as, for example, alkalinephosphatase promoter, tryptophan (trp) promoter, lambda P_(L) promoter,β-lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7promoter, or tac promoter (de Boer et al., 1983).

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,a sequence encoding a signal peptide, and/or enhancer elements.Transcription termination regions can typically be obtained from the 3′untranslated region of a eukaryotic or viral gene sequence.Transcription termination sequences can be positioned downstream of acoding sequence to provide for efficient termination. A signal peptidesequence is a short amino acid sequence typically present at the aminoterminus of a protein that is responsible for the relocation of anoperably linked mature polypeptide to a wide range of post-translationalcellular destinations, ranging from a specific organelle compartment tosites of protein action and the extracellular environment. Targetinggene products to an intended cellular and/or extracellular destinationthrough the use of an operably linked signal peptide sequence iscontemplated for use with the polypeptides of the invention. Classicalenhancers are cis-acting elements that increase gene transcription andcan also be included in the expression construct. Classical enhancerelements are known in the art, and include, but are not limited to, theCaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancerelement, and the SV40 enhancer element. Intron-mediated enhancerelements that enhance gene expression are also known in the art. Theseelements must be present within the transcribed region and areorientation dependent.

DNA sequences which direct polyadenylation of mRNA transcribed from theexpression construct can also be included in the expression construct,and include, but are not limited to, an octopine synthase or nopalinesynthase signal.

Expression constructs can also include one or more dominant selectablemarker genes, including, for example, genes encoding antibioticresistance and/or herbicide-resistance for selecting transformed cells.Antibiotic-resistance genes can provide for resistance to one or more ofthe following antibiotics: hygromycin, kanamycin, bleomycin, G418,streptomycin, paromomycin, neomycin, and spectinomycin. Kanamycinresistance can be provided by neomycin phosphotransferase (NPT II).Herbicide-resistance genes can provide for resistance tophosphinothricin acetyltransferase or glyphosate. Other markers used forcell transformation screening include, but are not limited to, genesencoding β-glucuronidase (GUS), β-galactosidase, luciferase, nopalinesynthase, chloramphenicol acetyltransferase (CAT), green fluorescenceprotein (GFP), or enhanced GFP (Yang et al., 1996).

The subject invention also concerns polynucleotide vectors comprising apolynucleotide sequence of the invention that encodes a polypeptide ofthe invention. Unique restriction enzyme sites can be included at the 5′and 3′ ends of an expression construct or polynucleotide of theinvention to allow for insertion into a polynucleotide vector. As usedherein, the term “vector” refers to any genetic element, including forexample, plasmids, cosmids, chromosomes, phage, virus, and the like,which is capable of replication when associated with proper controlelements and which can transfer polynucleotide sequences between cells.Vectors contain a nucleotide sequence that permits the vector toreplicate in a selected host cell. A number of vectors are available forexpression and/or cloning, and include, but are not limited to, pBR322,pUC series, M13 series, pGEM series, and pBLUESCRIPT vectors(Stratagene, La Jolla, Calif. and Promega, Madison, Wis.).

The subject invention also concerns oligonucleotide probes and primers,such as polymerase chain reaction (PCR) primers, that can hybridize to acoding or non-coding sequence of a polynucleotide of the presentinvention. Oligonucleotide probes of the invention can be used inmethods for detecting influenza virus nucleic acid sequences.Oligonucleotide primers of the invention can be used in PCR methods andother methods involving nucleic acid amplification. In a preferredembodiment, a probe or primer of the invention can hybridize to apolynucleotide of the invention under stringent conditions. Probes andprimers of the invention can optionally comprise a detectable label orreporter molecule, such as fluorescent molecules, enzymes, radioactivemoiety, and the like. Probes and primers of the invention can be of anysuitable length for the method or assay in which they are beingemployed. Typically, probes and primers of the invention will be 10 to500 or more nucleotides in length. Probes and primers that are 10 to 20,21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91to 100, or 101 or more nucleotides in length are contemplated within thescope of the invention. In one embodiment, probes and primers are any of9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides in length. Probes and primers of theinvention can have complete (100%) nucleotide sequence identity with thepolynucleotide sequence, or the sequence identity can be less than 100%.For example, sequence identity between a probe or primer and a sequencecan be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any otherpercentage sequence identity so long as the probe or primer canhybridize under stringent conditions to a nucleotide sequence of apolynucleotide of the invention. Exemplified probes and primers of theinvention include those having the nucleotide sequence shown in any ofSEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, or a functional fragment orvariant of any of the SEQ ID NOs: 35-46.

As used herein, the terms “nucleic acid,” “polynucleotide,” and“oligonucleotide” refer to a deoxyribonucleotide, ribonucleotide, or amixed deoxyribonucleotide and ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, would encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally-occurring nucleotides. Polynucleotide sequencesinclude the DNA strand sequence that can be transcribed into RNA and theRNA strand that can be translated into protein. The complementarysequence of any nucleic acid, polynucleotide, or oligonucleotide of thepresent invention is also contemplated within the scope of theinvention. Polynucleotide sequences also include both full-lengthsequences as well as shorter sequences derived from the full-lengthsequences. The subject invention also encompasses those polynucleotidesthat are complementary in sequence to the polynucleotides disclosedherein. Polynucleotides and polypeptides of the invention can beprovided in purified or isolated form.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode a polypeptide of the presentinvention. A table showing all possible triplet codons (and where U alsostands for T) and the amino acid encoded by each codon is described inLewin (1985). In addition, it is well within the skill of a persontrained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, polypeptides of the subjectinvention. These degenerate variant and alternative polynucleotidesequences are within the scope of the subject invention. As used herein,references to “essentially the same” sequence refers to sequences whichencode amino acid substitutions, deletions, additions, or insertionswhich do not materially alter the functional and/or immunogenic activityof the polypeptide encoded by the polynucleotides of the presentinvention.

The subject invention also concerns variants of the polynucleotides ofthe present invention that encode polypeptides of the invention. Variantsequences include those sequences wherein one or more nucleotides of thesequence have been substituted, deleted, and/or inserted. Thenucleotides that can be substituted for natural nucleotides of DNA havea base moiety that can include, but is not limited to, inosine,5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine,5-methylcytosine, and tritylated bases. The sugar moiety of thenucleotide in a sequence can also be modified and includes, but is notlimited to, arabinose, xylulose, and hexose. In addition, the adenine,cytosine, guanine, thymine, and uracil bases of the nucleotides can bemodified with acetyl, methyl, and/or thio groups. Sequences containingnucleotide substitutions, deletions, and/or insertions can be preparedand tested using standard techniques known in the art.

Substitution of amino acids other than those specifically exemplified ornaturally present in a polypeptide of the invention are alsocontemplated within the scope of the present invention. For example,non-natural amino acids can be substituted for the amino acids of apolypeptide, so long as the polypeptide having the substituted aminoacids retains substantially the same functional activity as thepolypeptide in which amino acids have not been substituted. Examples ofnon-natural amino acids include, but are not limited to, ornithine,citrulline, hydroxyproline, homoserine, phenylglycine, taurine,iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-aminohexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline,cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogues in general. Non-natural amino acids alsoinclude amino acids having derivatized side groups. Furthermore, any ofthe amino acids in the protein can be of the D (dextrorotary) form or L(levorotary) form. Allelic variants of a protein sequence of apolypeptide of the present invention are also encompassed within thescope of the invention.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a polypeptide of the present invention having anamino acid of one class is replaced with another amino acid of the sameclass fall within the scope of the subject invention so long as thepolypeptide having the substitution still retains substantially the samefunctional activity as the polypeptide that does not have thesubstitution. Polynucleotides encoding a polypeptide having one or moreamino acid substitutions in the sequence are contemplated within thescope of the present invention. Table 11 below provides a listing ofexamples of amino acids belonging to each class. Single letter aminoacid abbreviations are defined in Table 12.

Fragments and variants of polypeptides of influenza virus of the presentinvention can be generated using standard methods known in the art andtested for the presence of function or immunogenecity using standardtechniques known in the art. For example, for testing fragments and/orvariants of a neuraminidase polypeptide of the invention, enzymaticactivity can be assayed. Thus, an ordinarily skilled artisan can readilyprepare and test fragments and variants of a polypeptide of theinvention and determine whether the fragment or variant retains activityrelative to full-length or a non-variant polypeptide.

Polynucleotides and polypeptides contemplated within the scope of thesubject invention can also be defined in terms of more particularidentity and/or similarity ranges with those sequences of the inventionspecifically exemplified herein. The sequence identity will typically begreater than 60%, preferably greater than 75%, more preferably greaterthan 80%, even more preferably greater than 90%, and can be greater than95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (1990), modified as in Karlinand Altschul (1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990). BLAST searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainsequences with the desired percent sequence identity. To obtain gappedalignments for comparison purposes, Gapped BLAST can be used asdescribed in Altschul et al. (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide moleculeshaving sequences which are sufficiently homologous with thepolynucleotide sequences exemplified herein so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis et al., 1982). As used herein, “stringent”conditions for hybridization refers to conditions wherein hybridizationis typically carried out overnight at 20-25 C below the meltingtemperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution,0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, isdescribed by the following formula (Beltz et al., 1983):

Tm=81.5C+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

The subject invention also concerns viral proteins and peptides encodedby the genes of an influenza virus of the present invention. In oneembodiment, the viral protein is a mature HA protein. In a specificembodiment, the mature HA protein comprises one or more of thefollowing: a serine at position 82; a leucine at position 221; athreonine at position 327; and/or a threonine at position 482. In anexemplified embodiment, the mature HA protein has an amino acid sequenceshown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional and/orimmunogenic fragment or variant of SEQ ID NO: 33 or SEQ ID NO: 34. Inanother embodiment, the viral protein is an NA protein, NS protein, PBprotein, PA protein, or MA protein. Viral proteins and peptides of theinvention can be used to generate antibodies that bind specifically tothe protein or peptide. Viral proteins and peptides of the presentinvention can also be used as immunogens and in vaccine compositions.

The subject invention also concerns compositions and methods forinducing an immune response against an influenza virus that is capableof infecting a susceptible host animal and causing respiratory disease.The invention can be used to induce an immune response against aninfluenza virus of any subtype in a susceptible host animal. Forexample, the influenza virus can be an HA subtype of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and an NA subtypeof N1, N2, N3, N4, N5, N6, N7, N8, or N9. In one embodiment, the HAsubtype is H3 or H5. In a further embodiment, the NA subtype is N7 orN8. In a specific embodiment, an immune response is induced against aninfluenza virus of subtype H3N8. In one embodiment, the host animal is acanid. Canines include wild, zoo, and domestic canines, such as wolves,coyotes, and foxes. Canines also include dogs, particularly domesticdogs, such as, for example, pure-bred and/or mongrel companion dogs,show dogs, working dogs, herding dogs, hunting dogs, guard dogs, policedogs, racing dogs, and/or laboratory dogs. In a specific embodiment, thehost animal is a domesticated dog, such as a greyhound. In oneembodiment, an animal is administered an effective amount of animmunogenic composition of the present invention sufficient to induce animmune response against an influenza virus of the invention. The immuneresponse can be a humoral and/or cellular immune response. In a specificembodiment, the immune response is a protective immune response that iscapable of preventing or minimizing viral infection in the immunizedhost animal for some period of time subsequent to the immunization.Thus, the subject invention also concerns vaccine compositions andmethods that can provide a vaccinated animal with a protective immuneresponse to a virus of the present invention.

As described herein, the vaccine or immunogenic compositions of thesubject invention may comprise cell-free whole virus, includingattenuated or inactivated virus, or portions of the virus, includingsubvirion particles (including “split vaccine” wherein a virion istreated to remove some or all viral lipids), viral proteins (includingindividual proteins and macromolecular complexes of multiple proteins),polypeptides, and peptides, as well as virus-infected cell lines, or acombination of any of these. Vaccine or immunogenic compositionscomprising virus-infected cell lines may comprise multiple cell lines,each infected with a different viral strain.

In one embodiment of the invention, a canine may be immunized with oneor more inactivated (i.e., killed) and/or live attenuated influenzavirus vaccines or vaccines comprising one or a multiplicity of influenzavirus antigens from one or more virus isolates. In one embodiment, theinfluenza virus is a canine influenza virus. In another embodiment, theinfluenza virus is an equine influenza virus that encodes or expresses apolypeptide that has at least about 90%, or at least about 95%, or atleast about 96%, or 97%, or 98%, or 99% or more amino acid sequenceidentity with a canine influenza virus polypeptide. In one embodiment,an influenza antigen used in a vaccine of the present invention has atleast about 96% sequence identity with an HA antigen and/or NA antigenof a canine influenza virus.

An example of an inactivated vaccine is EQUICINE II™, which has beenmarketed by Intervet Inc. (Millsboro, Del., USA) as a liquid vaccine.EQUICINE II™ contains inactivated A/Pennsylvania/63 influenza virus(“A/Pa/63”) and A/equine/Kentucky/93 influenza virus (“A/KY/93”) withcarbopol (i.e., HAVLOGEN® (Intervet Inc.)). More specifically, a dose ofEQUICINE II™ contains: inactivated A/Pa/63 at 10^(6.0) EID₅₀,inactivated A/KY/93 at 10^(6.7) EID₅₀, 0.25% by volume carbopol, andsufficient PBS to create a total volume of 1 ml.

Another example of an inactivated vaccine is equine flu virusA/equine/Ohio/03 (“Ohio 03”). In some embodiments, such a vaccinecontains CARBIGEN™, which is an emulsified polymer-based adjuvantcommercially available from MVP Laboratories, Inc. (Ralston, Nebr.). Insuch vaccines, a dosage unit typically comprises at least about 250 HAunits of the virus, from about 250 to about 12,500 HA units of thevirus, or from about 1000 to about 6200 HA units of the virus. Therecommended concentration of CARBIGEN™ is from about 5 to about 30% (bymass).

An example of a live attenuated vaccine is modified liveequine/Kentucky/91 (“A/KY/91”) influenza in the form of a freeze-driedvaccine that may be reconstituted with water. In some embodiments, thisreconstitution is conducted using vaccine-grade water sufficient tobring the vaccine dosage to a total volume of 1 ml. Aspects of suchvaccines are discussed in, for example, U.S. Pat. Nos. 6,436,408;6,398,774; and 6,177,082, which are incorporated by reference in theirentirety into this patent. When reconstituted, a dose of such a vaccinemay, for example, contain A/KY/91 at 10^(7.2) TCID₅₀ per ml, 0.015 gramsN-Z AMINE AS™ per ml, 0.0025 grams gelatin per ml, and 0.04 grams Dlactose per ml. N-Z AMINE AS™ is a refined source of amino acids andpeptides produced by enzymatic hydrolysis of casein. N-Z AMINE AS™ ismarketed by Kerry Bio-Science (Norwich, N.Y., USA).

In a preferred embodiment, the vaccine comprises an H3 influenza antigenhaving at least about 93% homology with Florida/43/2004 in HA codingsequences, such as, for example, the equine/New Market/79 strain.Preferred homology is at least about 96%, such as, for example, theequine/Alaska/1/91 and equine/Santiago/85 strains. In the examples thatfollow, the equine/Kentucky/91, equine-2/Kentucky/93,equine-1/Pennsylvania/63, and equine Ohio/03 influenza antigens wereincorporated into vaccines. Preferred vaccines also include vaccinescomprising equine/Wisconsin/03, equine/Kentucky/02, equine/Kentucky/93,and equine/New Market 2/93. In the examples that follow, H3N8 viruseswere used. It is believed, however, that other H3 influenza viruses canbe used in accordance with this invention.

Live attenuated vaccines can be prepared by conventional means. Suchmeans generally include, for example, modifying pathogenic strains by invitro passaging, cold adaptation, modifying the pathogenicity of theorganism by genetic manipulation, preparation of chimeras, insertion ofantigens into viral vectors, selecting non-virulent wild type strains,etc.

In some embodiments, the live attenuated virus strain is derived byserial passage of the wild-type virus through cell culture, laboratoryanimals, non-host animals, or eggs. The accumulation of genetic mutationduring such passage(s) typically leads to progressive loss of virulenceof the organism to the original host.

In some embodiments, the live attenuated virus strain is prepared byco-infection of permissible cells with an attenuated mutant virus andpathogenic virus. The desired resultant recombinant virus has the safetyof the attenuated virus with genes coding for protective antigens fromthe pathogenic virus.

In some embodiments, the live attenuated virus strain is prepared bycold adaptation. A cold-adapted virus has an advantage of replicatingonly at the temperature found in upper respiratory tract. A method ofgeneration of a cold-adapted equine influenza virus has been describedin U.S. Pat. No. 6,177,082. A desired resulting cold-adapted virusconfers one or more of the following phenotypes: cold adaptation,temperature sensitivity, dominant interference, and/or attenuation.

In some embodiments, the live attenuated virus strain is prepared bymolecular means, such as point mutation, deletion, or insertion toconvert a pathogenic virus to a non-pathogenic or less-pathogenic viruscompared to the original virus, while preserving the protectiveproperties of the original virus.

In some embodiments, the live attenuated virus is prepared by cloningthe candidate of genes of protective antigens into a genome of anon-pathogenic or less-pathogenic virus or other organism.

Inactivated (i.e., “killed”) virus vaccines may be prepared byinactivating the virus using conventional methods. Typically, suchvaccines include excipients that may enhance an immune response, as wellas other excipients that are conventionally used in vaccines. Forexample, in the examples that follow, EQUICINE II™ comprises HAVLOGEN®.Inactivation of the virus can be accomplished by treating the virus withinactivation chemicals (e.g., formalin, beta propiolactone (“BPL”),bromoethylamine (“BEA”), and binary ethylenimine (“BEI”)) or bynon-chemical methods (e.g., heat, freeze/thaw, or sonication) to disablethe replication capacity of the virus.

In the examples that follow, equine/Ohio/03 was used as a challengevirus. It is known to have about 99% homology with Florida/43/04isolates, and has been shown to induce symptoms of infection andseroconversion in dogs. Example 18 illustrates the efficacy of equineinfluenza vaccine in dogs, showing hemagglutination inhibition (or “HI”or “HAI”) titers in dogs vaccinated with inactivated Ohio 03 antigen ina vaccine composition comprising CARBIGEN™ adjuvant. Table 29 showstiters pre-vaccination, post-vaccination, and post-second vaccination,as well as post-challenge. The results indicate HI titers at each stagepost-vaccination for the vaccinated dogs, with little or no increase forcontrols. Table 30 illustrates the clinical signs, virus isolation,

and histopathology results from the same study. Although challengedanimals did not show clinical signs, virus shedding, or positivehistopathology, the positive HI titers (Table 29) indicate significantantibody titers in immunized animals.

It should be noted that other H3 influenza virus antigen vaccines areencompassed by this invention as well. Those described in thisspecification and the following examples are provided to illustrate theinvention and its preferred embodiments, and not to limit the scope ofthe invention claimed.

It should further be noted that influenza antigens other than H3influenza virus antigens may be used in accordance with this invention.Such antigens include, for example, those from equine/PA/63, which is anequine A1 subtype (H7N7). It is contemplated that one or more of suchantigens may be used with or without one or more H3 influenza antigens.

In general, the vaccine is administered in a therapeutically effectiveamount. A “therapeutically effective amount” is an amount sufficient toinduce a protective response in the canine patient against the targetvirus. Typically, a dosage is “therapeutically effective” if itprevents, reduces the risk of, delays the onset of, reduces the spreadof, ameliorates, suppresses, or eradicates the influenza or one or more(typically two or more) of its symptoms. Typical influenza symptomsinclude, for example, fever (for dogs, typically ≧103.0° F.; ≧39.4° C.),cough, sneezing, histopathological lesions, ocular discharge, nasaldischarge, vomiting, diarrhea, depression, weight loss, gagging,hemoptysis, and/or audible rales. Other often more severe symptoms mayinclude, for example, hemorrhage in the lungs, mediastanum, or pleuralcavity; tracheitis; bronchitis; bronchiolitis; supportivebronchopneumonia; and/or infiltration of the epithelial lining andairway lumens of the lungs with neutrophils and/or macrophages.

The vaccine may be administered as part of a combination therapy, i.e.,a therapy that includes, in addition to the vaccine itself,administering one or more additional active agents, adjuvants,therapies, etc. In that instance, it should be recognized the amount ofvaccine that constitutes a “therapeutically effective” amount may beless than the amount of vaccine that would constitute a “therapeuticallyeffective” amount if the vaccine were to be administered alone. Othertherapies may include those known in the art, such as, for example,anti-viral medications, analgesics, fever-reducing medications,expectorants, anti-inflammation medications, antihistamines, antibioticsto treat bacterial infection that results from the influenza virusinfection, rest, and/or administration of fluids. In some embodiments,the vaccine of this invention is administered in combination with abordetella vaccine, adenovirus vaccine, and/or parainfluenza virusvaccine.

In some embodiments, for example, a typical dose for a live attenuatedvaccine is at least about 10³ pfu/canine, and more typically from about10³ to about 10⁹ pfu/canine. In this patent, “pfu” means “plaque formingunits”. In some embodiments, a typical dose for a live attenuatedvaccine is at least about 10³ TCID₅₀/canine, and more typically fromabout 10³ to about 10⁹ TCID₅₀/canine. In some embodiments, a typicaldose for a live attenuated vaccine is at least about 10³ EID₅₀/canine,and more typically from about 10³ to about 10⁹ EID₅₀/canine. In someembodiments, a typical dose for a killed vaccine is at least about 40 HAunits, typically from about 40 to about 10,000 HA units, and moretypically from about 500 to about 6200 HA units. In some embodiments,the dose is from about 6100 to about 6200 HA units.

In some preferred embodiments, the vaccine comprises a live attenuatedvaccine at a concentration which is at least about 10^(0.5) pfu/caninegreater than the immunogenicity level.

In some preferred embodiments, the vaccine comprises a live attenuatedvaccine at a concentration which is at least about 10^(0.5)TCID₅₀/canine greater than the immunogenicity level. In some preferredembodiments, the vaccine comprises a live attenuated vaccine at aconcentration which is at least about 10^(0.5) EID₅₀/canine greater thanthe immunogenicity level.

The immunogenicity level may be determined experimentally by challengedose titration study techniques generally known in the art. Suchtechniques typically include vaccinating a number of canines with thevaccine at different dosages, and then challenging the canines with thevirulent virus to determine the minimum protective dose.

Factors affecting the preferred dosage regimen may include, for example,the type (e.g., species and breed), age, weight, sex, diet, activity,lung size, and condition of the subject; the route of administration;the efficacy, safety, and duration-of-immunity profiles of theparticular vaccine used; whether a delivery system is used; and whetherthe vaccine is administered as part of a drug and/or vaccinecombination. Thus, the dosage actually employed can vary for specificanimals, and, therefore, can deviate from the typical dosages set forthabove. Determining such dosage adjustments is generally within the skillof those in the art using conventional means. It should further be notedthat live attenuated viruses are generally self-propagating; thus, thespecific amount of such a virus administered is not necessarilycritical.

It is contemplated that the vaccine may be administered to the caninepatient a single time; or, alternatively, two or more times over days,weeks, months, or years. In some embodiments, the vaccine isadministered at least two times. In some such embodiments, for example,the vaccine is administered twice, with the second dose (e.g., thebooster) being administered at least about 2 weeks after the first. Insome embodiments, the vaccine is administered twice, with the seconddose being administered no greater than 8 weeks after the first. In someembodiments, the second dose is administered at from about 2 weeks toabout 4 years after the first dose, from about 2 to about 8 weeks afterthe first dose, or from about 3 to about 4 weeks after the first dose.In some embodiments, the second dose is administered about 4 weeks afterthe first dose. In the above embodiments, the first and subsequentdosages may vary, such as, for example, in amount and/or form. Often,however, the dosages are the same as to amount and form. When only asingle dose is administered, the amount of vaccine in that dose alonegenerally comprises a therapeutically effective amount of the vaccine.When, however, more than one dose is administered, the amounts ofvaccine in those doses together may constitute a therapeuticallyeffective amount.

In some embodiments, the vaccine is administered before the caninerecipient is infected with influenza. In such embodiments, the vaccinemay, for example, be administered to prevent, reduce the risk of, ordelay the onset of influenza or one or more (typically two or more)influenza symptoms.

In some embodiments, the vaccine is administered after the caninerecipient is infected with influenza. In such embodiments, the vaccinemay, for example, ameliorate, suppress, or eradicate the influenza orone or more (typically two or more) influenza symptoms.

The preferred composition of the vaccine depends on, for example,whether the vaccine is an inactivated vaccine, live attenuated vaccine,or both. It also depends on the method of administration of the vaccine.It is contemplated that the vaccine will comprise one or moreconventional pharmaceutically acceptable carriers, adjuvants, otherimmune-response enhancers, and/or vehicles (collectively referred to as“excipients”). Such excipients are generally selected to be compatiblewith the active ingredient(s) in the vaccine. Use of excipients isgenerally known to those skilled in the art.

The term “pharmaceutically acceptable” is used adjectivally to mean thatthe modified noun is appropriate for use in a pharmaceutical product.When it is used, for example, to describe an excipient in apharmaceutical vaccine, it characterizes the excipient as beingcompatible with the other ingredients of the composition and notdisadvantageously deleterious to the intended recipient canine.

The vaccines may be administered by conventional means, including, forexample, mucosal administration, (such as intranasal, oral,intratracheal, and ocular), and parenteral administration. Mucosaladministration is often particularly advantageous for live attenuatedvaccines. Parenteral administration is often particularly advantageousfor inactivated vaccines.

Mucosal vaccines may be, for example, liquid dosage forms, such aspharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. Excipients suitable for such vaccines include, for example,inert diluents commonly used in the art, such as, water, saline,dextrose, glycerol, lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol. Excipients also cancomprise various wetting, emulsifying, suspending, flavoring (e.g.,sweetening), and/or perfuming agents.

Oral mucosal vaccines also may, for example, be tableted or encapsulatedfor convenient administration. Such capsules or tablets can contain acontrolled-release formulation. In the case of capsules, tablets, andpills, the dosage forms also can comprise buffering agents, such assodium citrate, or magnesium or calcium carbonate or bicarbonate.Tablets and pills additionally can be prepared with enteric coatings.

It is contemplated that the vaccine may be administered via the caninepatient's drinking water and/or food. It is further contemplated thatthe vaccine may be administered in the form of a treat or toy.

“Parenteral administration” includes subcutaneous injections, submucosalinjections, intravenous injections, intramuscular injections,intrasternal injections, transcutaneous injections, and infusion.Injectable preparations (e.g., sterile injectable aqueous or oleaginoussuspensions) can be formulated according to the known art using suitableexcipients, such as vehicles, solvents, dispersing, wetting agents,emulsifying agents, and/or suspending agents. These typically include,for example, water, saline, dextrose, glycerol, ethanol, corn oil,cottonseed oil, peanut oil, sesame oil, benzyl alcohol, benzyl alcohol,1,3-butanediol, Ringer's solution, isotonic sodium chloride solution,bland fixed oils (e.g., synthetic mono- or diglycerides), fatty acids(e.g., oleic acid), dimethyl acetamide, surfactants (e.g., ionic andnon-ionic detergents), propylene glycol, and/or polyethylene glycols.Excipients also may include small amounts of other auxiliary substances,such as pH buffering agents.

The vaccine may include one or more excipients that enhance a caninepatient's immune response (which may include an antibody response,cellular response, or both), thereby increasing the effectiveness of thevaccine. Use of such excipients (or “adjuvants”) may be particularlybeneficial when using an inactivated vaccine. The adjuvant(s) may be asubstance that has a direct (e.g., cytokine or Bacille Calmette-Guerin(“BCG”)) or indirect effect (liposomes) on cells of the canine patient'simmune system. Examples of often suitable adjuvants include oils (e.g.,mineral oils), metallic salts (e.g., aluminum hydroxide or aluminumphosphate), bacterial components (e.g., bacterial liposaccharides,Freund's adjuvants, and/or MDP), plant components (e.g., Quil A), and/orone or more substances that have a carrier effect (e.g., bentonite,latex particles, liposomes, and/or Quil A, ISCOM). As noted above,adjuvants also include, for example, CARBIGEN™ and carbopol. It shouldbe recognized that this invention encompasses both vaccines thatcomprise an adjuvant(s), as well as vaccines that do not comprise anyadjuvant.

It is contemplated that the vaccine may be freeze-dried (or otherwisereduced in liquid volume) for storage, and then reconstituted in aliquid before or at the time of administration. Such reconstitution maybe achieved using, for example, vaccine-grade water.

The present invention further comprises kits that are suitable for usein performing the methods described above. The kit comprises a dosageform comprising a vaccine described above. The kit also comprises atleast one additional component, and, typically, instructions for usingthe vaccine with the additional component(s). The additionalcomponent(s) may, for example, be one or more additional ingredients(such as, for example, one or more of the excipients discussed above,food, and/or a treat) that can be mixed with the vaccine before orduring administration. The additional component(s) may alternatively (oradditionally) comprise one or more apparatuses for administering thevaccine to the canine patient. Such an apparatus may be, for example, asyringe, inhaler, nebulizer, pipette, forceps, or any medicallyacceptable delivery vehicle. In some embodiments, the apparatus issuitable for subcutaneous administration of the vaccine. In someembodiments, the apparatus is suitable for intranasal administration ofthe vaccine.

Other excipients and modes of administration known in the pharmaceuticalor biologics arts also may be used.

The vaccine or immunogenic compositions of the subject invention alsoencompass recombinant viral vector-based constructs that may comprise,for example, genes encoding HA protein, NA protein, nucleoprotein,polymerase basic protein, polymerase acidic protein, and/or matrixprotein of an influenza virus of the present invention. Any suitableviral vector that can be used to prepare a recombinant vector/virusconstruct is contemplated for use with the subject invention. Forexample, viral vectors derived from adenovirus, avipox, herpesvirus,vaccinia, canarypox, entomopox, swinepox, West Nile virus and othersknown in the art can be used with the compositions and methods of thepresent invention. Recombinant polynucleotide vectors that encode andexpress components can be constructed using standard genetic engineeringtechniques known in the art. In addition, the various vaccinecompositions described herein can be used separately and in combinationwith each other. For example, primary immunizations of an animal may userecombinant vector-based constructs, having single or multiple straincomponents, followed by secondary boosts with vaccine compositionscomprising inactivated virus or inactivated virus-infected cell lines.Other immunization protocols with the vaccine compositions of theinvention are apparent to persons skilled in the art and arecontemplated within the scope of the present invention.

The subject invention also concerns reassortant virus comprising atleast one gene or genomic segment of an influenza virus of the presentinvention and the remainder of viral genes or genomic segments from adifferent influenza virus of the invention or from an influenza virusother than a virus of the present invention. Reassortant virus can beproduced by genetic reassortant of nucleic acid of a donor influenzavirus of the present invention with nucleic acid of a recipientinfluenza virus and then selecting for reassortant virus that comprisesthe nucleic acid of the donor virus. Methods to produce and isolatereassortant virus are well known in the art (Fields et al., 1996). Inone embodiment, a reassortant virus of the invention comprises genes orgenomic segments of a human, avian, swine, or equine influenza virus. Areassortant virus of the present invention can include any combinationof nucleic acid from donor and recipient influenza virus so long as thereassortant virus comprises at least one gene or genomic segment from adonor influenza virus of the present invention. In one embodiment, arecipient influenza virus can be an equine influenza virus.

Natural, recombinant or synthetic polypeptides of viral proteins, andpeptide fragments thereof, can also be used as vaccine compositionsaccording to the subject methods. In one embodiment, a vaccinecomposition comprises a polynucleotide or a polypeptide of a canineinfluenza virus. In one embodiment, a vaccine composition comprises apolynucleotide encoding a polypeptide having the amino acid sequenceshown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, or 78, or a functional and/or immunogenic fragment orvariant thereof. In a specific embodiment, the polynucleotide encodingthe amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, or 78, comprises the nucleotide sequenceshown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or77, respectively, or a sequence encoding a functional and/or immunogenicfragment or variant of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, or 78. In a further specific embodiment, apolynucleotide of the invention can comprise: Nucleotides 1-2271 of SEQID NO: 3; Nucleotides 1-2148 of SEQ ID NO: 5; Nucleotides 1-657 of SEQID NO: 7; Nucleotides 1-1494 of SEQ ID NO: 9; Nucleotides 1-1410 of SEQID NO: 11; Nucleotides 1-756 of SEQ ID NO: 13; Nucleotides 1-1695 of SEQID NO: 15; Nucleotides 1-2271 of SEQ ID NO: 19; Nucleotides 1-2148 ofSEQ ID NO: 21; Nucleotides 1-657 of SEQ ID NO: 23; Nucleotides 1-1494 ofSEQ ID NO: 25; Nucleotides 1-756 of SEQ ID NO: 29; Nucleotides 1-1695 ofSEQ ID NO: 31; Nucleotides 1-2277 of SEQ ID NO: 47; Nucleotides 1-2271of SEQ ID NO: 49; Nucleotides 1-2148 of SEQ ID NO: 51; Nucleotides 1-690of SEQ ID NO: 53; Nucleotides 1-1494 of SEQ ID NO: 55; Nucleotides1-1410 of SEQ ID NO: 57; Nucleotides 1-756 of SEQ ID NO: 59; Nucleotides1-1695 of SEQ ID NO: 61; Nucleotides 1-2277 of SEQ ID NO: 63;Nucleotides 1-2271 of SEQ ID NO: 65; Nucleotides 1-2148 of SEQ ID NO:67; Nucleotides 1-690 of SEQ ID NO: 69; Nucleotides 1-1494 of SEQ ID NO:71; Nucleotides 1-1410 of SEQ ID NO: 73; Nucleotides 1-756 of SEQ ID NO:75; and Nucleotides 1-1695 of SEQ ID NO: 77. In another embodiment, avaccine composition comprises a polypeptide having the amino acidsequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, or 78, or a functional and/or immunogenic fragmentor variant thereof. In a further embodiment, a vaccine compositioncomprises a polynucleotide or a polypeptide of an equine influenza viruswherein the polynucleotide or polypeptide has at least about 90%, or atleast about 95%, or at least about 96%, or 97%, or 98%, or 99% or moresequence identity with a canine influenza polynucleotide or polypeptide.In one embodiment, viral polypeptides derived from multiple strains canbe combined in a vaccine composition and are used to vaccinate a hostanimal. For example, polypeptides based on the viral HA protein from atleast two different strains of influenza virus of the invention can becombined in the vaccine. The polypeptides may be homologous to onestrain or may comprise Ahybrid@ or Achimeric@ polypeptides whose aminoacid sequence is derived from joining or linking polypeptides from atleast two distinct strains. Procedures for preparing viral polypeptidesare well known in the art. For example, viral polypeptides and peptidescan be synthesized using solid-phase synthesis methods (Merrifield,1963). Viral polypeptides and peptides can also be produced usingrecombinant DNA techniques wherein a polynucleotide molecule encoding anviral protein or peptide is expressed in a host cell, such as bacteria,yeast, or mammalian cell lines, and the expressed protein purified usingstandard techniques of the art.

Vaccine compositions of the present invention also include naked nucleicacid compositions. In one embodiment, a nucleic acid may comprise anucleotide sequence encoding an HA and/or an NA protein of an influenzavirus of the present invention. Methods for nucleic acid vaccination areknown in the art and are described, for example, in U.S. Pat. Nos.6,063,385 and 6,472,375. The nucleic acid can be in the form of aplasmid or a gene expression cassette. In one embodiment, the nucleicacid is provided encapsulated in a liposome which is administered to ananimal.

Vaccine compositions and immunogens, such as polypeptides and nucleicacids, that can be used in accordance with the present invention can beprovided with a pharmaceutically-acceptable carrier or diluent.Compounds and compositions useful in the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W.Martin, Easton Pa., Mack Publishing Company, 19^(th) ed., 1995,describes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of an immunogen is combinedwith a suitable carrier in order to facilitate effective administrationof the composition. The compositions used in the present methods canalso be in a variety of forms. These include, for example, solid,semi-solid, and liquid dosage forms, such as tablets, pills, powders,liquid solutions or suspension, suppositories, injectable and infusiblesolutions, and sprays. The preferred form depends on the intended modeof administration and therapeutic application. The compositions alsopreferably include conventional pharmaceutically acceptable carriers anddiluents which are known to those skilled in the art. Examples ofcarriers or diluents for use with the subject peptidomimetics include,but are not limited to, water, saline, oils including mineral oil,ethanol, dimethyl sulfoxide, gelatin, cyclodextrans, magnesium stearate,dextrose, cellulose, sugars, calcium carbonate, glycerol, alumina,starch, and equivalent carriers and diluents, or mixtures of any ofthese. Formulations of an immunogen of the invention can also comprisesuspension agents, protectants, lubricants, buffers, preservatives, andstabilizers. To provide for the administration of such dosages for thedesired therapeutic treatment, pharmaceutical compositions of theinvention will advantageously comprise between about 0.1% and 45%, andespecially, 1 and 15% by weight of the immunogen or immunogens based onthe weight of the total composition including carrier or diluent.

The vaccine and immunogenic compositions of the subject invention can beprepared by procedures well known in the art. For example, the vaccineor immunogens are typically prepared as injectables, e.g., liquidsolutions or suspensions. The vaccine or immunogens are administered ina manner that is compatible with dosage formulation, and in such amountas will be therapeutically effective and immunogenic in the recipient.The optimal dosages and administration patterns for a particular vaccineor immunogens formulation can be readily determined by a person skilledin the art.

Peptides and/or polypeptides of the present invention can also beprovided in the form of a multiple antigenic peptide (MAP) construct.The preparation of MAP constructs has been described in Tam (1988). MAPconstructs utilize a core matrix of lysine residues onto which multiplecopies of an immunogen are synthesized (Posnett et al., 1988). MultipleMAP constructs, each containing the same or different immunogens, can beprepared and administered in a vaccine composition in accordance withmethods of the present invention. In one embodiment, a MAP construct isprovided with and/or administered with one or more adjuvants. Influenzapolypeptides of the invention can also be produced and administered asmacromolecular protein structures comprising one or more polypeptides.Published U.S. Patent Application US2005/0009008 describes methods forproducing virus-like particles as a vaccine for influenza virus.

According to the methods of the subject invention, the vaccine andimmunogenic compositions described herein are administered tosusceptible hosts, typically canids, and more typically domesticateddogs, in an effective amount and manner to induce protective immunityagainst subsequent challenge or infection of the host by virus. In oneembodiment, the host animal is a canid. Canines include wild, zoo, anddomestic canines, such as wolves, coyotes, and foxes. Canines alsoinclude dogs, particularly domestic dogs, such as, for example,pure-bred and/or mongrel companion dogs, show dogs, working dogs,herding dogs, hunting dogs, guard dogs, police dogs, racing dogs, and/orlaboratory dogs. In a specific embodiment, the host animal is adomesticated dog, such as a greyhound. The vaccines or immunogens aretypically administered parenterally, by injection, for example, eithersubcutaneously, intraperitoneally, or intramuscularly. Other suitablemodes of administration include oral or nasal administration. Usually,the vaccines or immunogens are administered to an animal at least twotimes, with an interval of one or more weeks between eachadministration. However, other regimens for the initial and boosteradministrations of the vaccine or immunogens are contemplated, and maydepend on the judgment of the practitioner and the particular hostanimal being treated.

Virus and virus-infected cells in a vaccine formulation may beinactivated or attenuated using methods known in the art. For example,whole virus and infected cells can be inactivated or attenuated byexposure to paraformaldehyde, formalin, beta propiolactone (BPL),bromoethylamine (BEA), binary ethylenimine (BEI), phenol, UV light,elevated temperature, freeze thawing, sonication (includingultrasonication), and the like. The amount of cell-free whole virus in avaccine dose can be in the range from about 0.1 mg to about 5 mg, andmore usually being from about 0.2 mg to about 2 mg. The dosage forvaccine formulations comprising virus-infected cell lines will usuallycontain from about 10⁶ to about 10⁸ cells per dose, and more usuallyfrom about 5×10⁶ to about 7.5×10⁷ cells per dose. The amount of proteinor peptide immunogen in a dose for an animal can vary from about 0.1 μgto 10000 μg, or about 1 μg to 5000 μg, or about 10 μg to 1000 μg, orabout 25 μg to 750 μg, or about 50 μg to 500 μg, or 100 μg to 250 μg,depending upon the size, age, etc., of the animal receiving the dose.

An immunogenic or vaccine composition of the invention, such as virus orvirus-infected cells or viral proteins or peptides, can be combined withan adjuvant, typically just prior to administration. Adjuvantscontemplated for use in the vaccine formulations include threonylmuramyl dipeptide (MDP) (Byars et al., 1987), saponin, Cornebacteriumparvum, Freund's complete and Fruend's incomplete adjuvants, aluminum,or a mixture of any of these. A variety of other adjuvants suitable foruse with the methods and vaccines of the subject invention, such asalum, are well known in the art and are contemplated for use with thesubject invention.

The subject invention also concerns antibodies that bind specifically toa protein or a peptide of the present invention. Antibodies of thesubject invention include monoclonal and polyclonal antibodycompositions. Preferably, the antibodies of the subject invention aremonoclonal antibodies. Whole antibodies and antigen binding fragmentsthereof are contemplated in the present invention. Thus, for example,suitable antigen binding fragments include Fab₂, Fab and Fv antibodyfragments. Antibodies of the invention can be labeled with a detectablemoiety, such as a fluorescent molecule (e.g., fluorescein or an enzyme).

The subject invention also concerns methods and compositions fordetection and identification of an influenza virus of the invention andfor diagnosis of infection of an animal with an influenza virus of thepresent invention. The methods of the invention include detection of thepresence of canine influenza, in a biological sample from an animal. Thedetection of canine influenza in a sample, is useful to diagnose canineinfluenza in an animal. In turn, this information can provide theability to determine the prognosis of an animal based on distinguishinglevels of canine influenza present over time, and can assist inselection of therapeutic agents and treatments for the animal, andassist in monitoring therapy. The method also provides the ability toestablish the absence of canine influenza in an animal tested.

The ability to detect canine influenza in an animal permits assessmentof outbreaks of canine influenza in different geographical locations.This information also permits early detection so that infected animalscan be isolated, to limit the spread of disease, and allows earlyintervention for treatment options. In addition, having this informationavailable can provide direction to medical personnel for preparing totreat large numbers of ill animals, including assembling medicalsupplies, and, if available, vaccines.

In one embodiment, a method of the present invention involves thecollection of a biological sample from a test animal, such as a canine.The biological sample may be any biological material, including, cells,tissue, hair, whole blood, serum, plasma, nipple aspirate, lung lavage,cerebrospinal fluid, saliva, sweat and tears.

The animal test sample may come from an animal suspected of havingcanine influenza virus, whether or not the animal exhibits symptoms ofthe disease. Control samples can also be provided or collected fromanimals known to be free of canine influenza. Additional controls may beprovided, e.g., to reduce false positive and false negative results, andverify that the reagents in the assay are actively detecting canineinfluenza A virus.

In addition to detecting the presence or absence of canine influenza ina biological sample, the methods of detection used in the invention candetect mutations in canine influenza virus, such as changes in nucleicacid sequence, that may result from the environment, drug treatment,genetic manipulations or mutations, injury, change in diet, aging, orany other characteristic(s) of an animal. Mutations may also causecanine influenza A to become resistant to a drug that was formerlyeffective, or to enable the virus to infect and propagate in a differentspecies of animal, or human. For example, avian influenza A virus hasbeen shown to infect other animals and humans.

In one embodiment for detecting an influenza virus in an animal,diagnosis is facilitated by the collection of high-quality specimens,their rapid transport to a testing facility, and appropriate storage,before laboratory testing. Virus is best detected in specimenscontaining infected cells and secretions. In one embodiment, specimensfor the direct detection of viral antigens and/or for nucleic acidsand/or virus isolation in cell cultures are taken during the first 3days after onset of clinical symptoms. A number of types of specimensare suitable to diagnose virus infections of the upper respiratorytract, including, but not limited to, nasal swab, nasopharyngeal swab,nasopharyngeal aspirate, nasal wash and throat swabs. In addition toswabs, samples of tissue or serum may be taken, and invasive procedurescan also be performed.

In one embodiment, respiratory specimens are collected and transportedin 1-5 ml of virus transport media. A number of media that aresatisfactory for the recovery of a wide variety of viruses arecommercially available. Clinical specimens are added to transportmedium. Nasal or nasopharyngeal swabs can also be transported in thevirus transport medium. One example of a transport medium is 10 gm ofveal infusion broth and 2 gm of bovine albumin fraction V, added tosterile distilled water to 400 m. Antibiotics such as 0.8 ml gentamicinsulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 μg/ml) canalso be added. The medium is preferably sterilized by filtration. Nasalwashes, such as sterile saline (0.85% NaCl), can also be used to collectspecimens of respiratory viruses.

In one embodiment, sera is collected in an amount of from 1-5 ml ofwhole blood from an acute-phase animal, soon after the onset of clinicalsymptoms, and preferably not later than 7 days. A convalescent-phaseserum specimen can also be collected, for example at about 14 days afteronset of symptoms. Serum specimens can be useful for detectingantibodies against respiratory viruses in a neutralization test.

In some instances, samples may be collected from individual animals overa period of time (e.g., once a day, once a week, once a month,biannually or annually). Obtaining numerous samples from an individualanimal, over a period of time, can be used to verify results fromearlier detections, and/or to identify response or resistance to aspecific treatment, e.g., a selected therapeutic drug.

The methods of the present invention can be used to detect the presenceof one or more pathological agents in a test sample from an animal, andthe level of each pathological agent. Any method for detecting thepathological agent can be used, including, but not limited to, antibodyassays including enzyme-linked immunosorbent assays (ELISAs), indirectfluorescent antibody (IFA) tests, hemagglutinating, and inhibition ofhemagglutination (HI) assays, and Western Blot. Known cell-culturemethods can also be used. Positive cultures can be further identifiedusing immunofluorescence of cell cultures or HI assay of the cellculture medium (supernatant).

In addition, methods for detecting nucleic acid (DNA or RNA) or proteincan be used. Such methods include, but are not limited to, polymerasechain reaction (PCR), and reverse transcriptase (RT) PCR tests and realtime tests, and quantitative nuclease protection assays. There arecommercially available test kits available to perform these assays. Forexample, QIAGEN (Valencia, Calif.) sells a one step RT-PCR kit, andviral RNA extraction kit.

In one embodiment, the method utilizes an antibody specific for a virusor viral protein of the invention. In a specific embodiment, an antibodyspecific for an HA protein of a virus of the invention is utilized. Inanother embodiment, an antibody specific for an NP protein of a virus ofthe invention is used. A suitable sample, such as from the nasal ornasopharyngeal region, is obtained from an animal and virus or viralprotein is isolated therefrom. The viral components are then screenedfor binding of an antibody specific to a protein, such as HA or NP, of avirus of the invention. In another embodiment, a serum sample (or otherantibody containing sample) is obtained from an animal and the serumscreened for the presence of antibody that binds to a protein of a virusof the invention. For example, an ELISA assay can be performed where theplate walls have HA and/or NP protein, or a peptide fragment thereof,bound to the wall. The plate wall is then contacted with serum orantibody from a test animal. The presence of antibody in the animal thatbinds specifically to the HA and/or NP protein is indicative that thetest animal is infected or has been infected with an influenza virus ofthe present invention.

In one embodiment, the presence of a pathological agent is detected bydetermining the presence or absence of antibodies against the agent, ina biological sample. It can take some time (e.g. months) after an animalis infected before antibodies can be detected in a blood test. Onceformed, antibodies usually persist for many years, even after successfultreatment of the disease. Finding antibodies to canine influenza A maynot indicate whether the infection was recent, or sometime in the past.

Antibody testing can also be done on fluid(s). Antibody assays includeenzyme-linked immunosorbent assays (ELISAs), indirect fluorescentantibody (IFA) assays, and Western Blot. Preferably, antibody testing isdone using multiple assays, for example ELISA or IFA followed by Westernblot. Antibody assays can be done in a two-step process, using either anELISA or IFA assay, followed by a Western blot assay. ELISA isconsidered a more reliable and accurate assay than IFA, but IFA may beused if ELISA is not available. The Western blot test (which is a morespecific test) can also be done in all animals, particularly those thathave tested positive or borderline positive (equivocal) in an ELISA orIFA assay.

Other antibody-based tests that can be used for detection of influenzavirus include hemagglutination inhibition assays. Hemagglutinationactivity can be detected in a biological sample from an animal, usingchicken or turkey red blood cells as described (Burleson et al., 1992)and Kendal et al., 1982). In one embodiment, an influenza or an HAprotein or peptide of the invention is contacted with a test samplecontaining serum or antibody. Red blood cells (RBC) from an animal, suchas a bird, are then added. If antibody to HA is present, then the RBCwill not agglutinate. If antibody to HA is not present, the RBC willagglutinate in the presence of HA. Variations and modifications tostandard hemagglutination inhibition assays are known in the art andcontemplated within the scope of the present invention.

Infection of an animal can also be determined by isolation of the virusfrom a sample, such as a nasal or nasopharyngeal swab. Viral isolationcan be performed using standard methods, including cell culture and egginoculation.

In a further embodiment, a nucleic acid-based assay can be used fordetection of a virus of the present invention. In one embodiment, anucleic acid sample is obtained from an animal and the nucleic acidsubjected to PCR using primers that will generate an amplificationproduct if the nucleic acid contains a sequence specific to an influenzavirus of the present invention. In a specific embodiment, RT-PCR is usedin an assay for the subject virus. In an exemplified embodiment,real-time RT-PCR is used to assay for an influenza virus of theinvention. PCR, RT-PCR and real-time PCR methods are known in the artand have been described in U.S. Pat. Nos. 4,683,202; 4,683,195;4,800,159; 4,965,188; 5,994,056; 6,814,934; and in Saiki et al. (1985);Sambrook et al. (1989); Lee et al. (1993); and Livak et al. (1995). Inone embodiment, the PCR assay uses oligonucleotides specific for aninfluenza matrix (MA) gene and/or HA gene. The amplification product canalso be sequenced to determine if the product has a sequence of aninfluenza virus of the present invention. Other nucleic acid-basedassays can be used for detection and diagnosis of viral infection by avirus of the invention and such assays are contemplated within the scopeof the present invention. In one embodiment, a sample containing anucleic acid is subjected to a PCR-based amplification using forward andreverse primers where the primers are specific for a viralpolynucleotide or gene sequence. If the nucleic acid in the sample isRNA, then RT-PCR can be performed. For real-time PCR, a detectable probeis utilized with the primers.

Primer sets specific for the hemagglutinin (HA) gene of many of thecirculating influenza viruses are known, and are continually beingdeveloped. The influenza virus genome is single-stranded RNA, and a DNAcopy (cDNA) must be made using a reverse transcriptase (RT) polymerase.The amplification of the RNA genome, for example using RT-PCR, requiresa pair of oligonucleotide primers, typically designed on the basis ofthe known HA sequence of influenza A subtypes and of neuraminadase(NM)-1. The primers can be selected such that they will specificallyamplify RNA of only one virus subtype. DNAs generated by usingsubtype-specific primers can be further analyzed by molecular genetictechniques such as sequencing. The test is preferably run with apositive control, or products are confirmed by sequencing and comparisonwith known sequences. The absence of the target PCR products (i.e, a“negative” result) may not rule out the presence of the virus. Resultscan then be made available within a few hours from either clinical swabsor infected cell cultures. PCR and RT-PCR tests for influenza A virusare described by Fouchier et al., 2000 and Maertzdorf et al., 2004.

The subject invention also concerns methods for screening for compoundsor drugs that have antiviral activity against a virus of the presentinvention. In one embodiment, cells infected with a virus of theinvention are contacted with a test compound or drug. The amount ofvirus or viral activity following contact is then determined. Thosecompounds or drugs that exhibit antiviral activity can be selected forfurther evaluation.

The subject invention also concerns isolated cells infected with aninfluenza virus of the present invention. In one embodiment, the cell isa canine cell, such as canine kidney epithelial cells.

The subject invention also concerns cells transformed with apolynucleotide of the present invention encoding a polypeptide of theinvention. Preferably, the polynucleotide sequence is provided in anexpression construct of the invention. More preferably, the expressionconstruct provides for overexpression in the cell of an operably linkedpolynucleotide of the invention. In one embodiment, the cell istransformed with a polynucleotide sequence comprising a sequenceencoding the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, or a functionalfragment or variant thereof. In a specific embodiment, the cell istransformed with a polynucleotide encoding the amino acid sequence shownin SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,or 78 comprises the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, respectively, or a sequenceencoding a functional fragment or variant of any of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78. Thus, the subjectinvention concerns cells transformed with a polynucleotide sequencecomprising the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, or a fragment or variant,including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, or 77.

The transformed cell can be a eukaryotic cell, for example, a plantcell, including protoplasts, or the transformed cell can be aprokaryotic cell, for example, a bacterial cell such as E. coli or B.subtilis. Animal cells include human cells, mammalian cells, partiallycanine cells, avian cells, and insect cells. Plant cells include, butare not limited to, dicotyledonous, monocotyledonous, and conifer cells.

The subject invention also concerns plants, including transgenic plantsthat express and produce a viral protein or polypeptide of the presentinvention. Plants, plant tissues, and plant cells transformed with orbred to contain a polynucleotide of the invention are contemplated bythe present invention. Preferably, the polynucleotide of the inventionis overexpressed in the plant, plant tissue, or plant cell. Plants canbe used to produce influenza vaccine compositions of the presentinvention and the vaccines can be administered through consumption ofthe plant (see, for example, U.S. Pat. Nos. 5,484,719 and 6,136,320).

The subject invention also concerns kits for detecting a virus ordiagnosing an infection by a virus of the present invention. In oneembodiment, a kit comprises an antibody of the invention thatspecifically binds to an influenza virus of the present invention, or anantigenic portion thereof. In another embodiment, a kit comprises one ormore polypeptides or peptides of the present invention. In a specificembodiment, the polypeptides have an amino acid sequence shown in any ofSEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or78, or a functional and/or immunogenic fragment or variant thereof. In afurther embodiment, a kit comprises one or more polynucleotides oroligonucleotides of the present invention. In a specific embodiment, thepolynucleotides have a nucleotide sequence shown in any of SEQ ID NOs.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77, or a fragment orvariant thereof. A kit may optionally comprise one or more controlantibody, control polypeptide or peptide, and/or control polynucleotideor oligonucleotide. The antibody, polypeptides, peptides,polynucleotides, and/or oligonucleotides of the kit can be provided in asuitable container or package.

The subject application also concerns the use of mongrel dogs as a modelfor infection and pathogenesis of influenza virus. In one embodiment, amongrel dog is inoculated with an influenza virus, such as a canineinfluenza virus of the present invention. Optionally, the dog can beadministered therapeutic agents subsequent to inoculation. The dog canalso have been administered a composition for generating an immuneresponse against an influenza virus prior to inoculation with virus.Tissue, blood, serum, and other biological samples can be obtainedbefore and/or after inoculation and examined for the presence of virusand pathogenesis of tissue using methods known in the art including, butnot limited to, PCR, RT-PCR, nucleic acid sequencing, andimmunohistochemistry.

Canine influenza virus strains (designated as “A/canine/Florida/43/2004”(ATCC Accession No. PTA-7914) and “A/canine/Florida/242/2003” (ATCCAccession No. PTA-7915)) were deposited with American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108, on Oct. 9, 2006.Canine influenza virus strains (designated as “canine/Jax/05” (ATCCAccession No. PTA 7941) and “canine/Miami/05” (ATCC Accession No.PTA-7940)), were deposited with American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108, on Oct. 17, 2006. The subject virusstrains have been deposited under conditions that assure that access tothe cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposit will be available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject virus deposits will be stored and made available tothe public in accord with the provisions of the Budapest Treaty for theDeposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

Table 57 illustrates the similarities among the amino acid sequencesencoded by the hemagglutinin (or “HA”), neuraminidase (or “NA”), andnucleoprotein (NP) genes of the canine influenza virus identified asA/canine/Florida/43/2004 (Ca/Fla/43/04) with H3N8 equine isolates, aswell as the canine/Florida/242/2003 isolate.

Any element of any embodiment disclosed herein can be combined with anyother element or embodiment disclosed herein and such combinations arespecifically contemplated within the scope of the present invention.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods for Examples 1-6

Blood and Nasal Swab Collection from Greyhounds.

Acute and convalescent blood samples were collected by jugularvenipuncture from clinically diseased or normal greyhounds in racingkennels experiencing outbreaks of respiratory disease. Convalescentsamples were collected 4 to 12 weeks after the acute sample. Serum washarvested and stored at −80° C. Nasal swabs were collected and placed inAmies transport medium with charcoal (Becton Dickinson Biosciences)pending submission for bacterial isolation.

Postmortem Examination of Greyhounds.

Complete postmortem examinations were performed by the AnatomicPathology Service at the University of Florida College of VeterinaryMedicine (UF CVM) on 5 of the 8 greyhounds that died in the January 2004outbreak at a Florida track. Postmortem examination of another dog wasperformed at a private veterinary clinic with submission of tissues tothe UF CVM for histopathologic diagnosis. Tissues were fixed in 10%neutral buffered formalin, embedded in paraffin, and 5-μm sections wereeither stained with hematoxylin and eosin for histopathologic diagnosisor processed for immunohistochemistry as described below. Unfixedtissues were submitted for bacterial culture and also stored at −80° C.

Serological Tests for Canine Viral Respiratory Pathogens.

Paired acute and convalescent serum samples were submitted to the AnimalHealth Diagnostic Laboratory (AHDL) at the Cornell University College ofVeterinary Medicine for serum neutralization assays against caninedistemper virus, adenovirus type 2, and parainfluenza virus. Antibodytiters were expressed as the last dilution of serum that inhibited viralinfection of cell cultures. Seroconversion, defined as a ≧4-foldincrease in antibody titer between the acute and convalescent sample,indicated viral infection. No seroconversions to these viral pathogenswere detected.

Microbial Tests for Canine Bacterial Respiratory Pathogens.

Paired nasal swabs and postmortem tissues were submitted to theDiagnostic Clinical Microbiology/Parasitology/Serology Service at the UFCVM for bacterial isolation and identification. The samples werecultured on nonselective media as well as media selective for Bordetellaspecies (Regan-Lowe; Remel) and Mycoplasma species (Remel). All cultureswere held for 21 days before reporting no growth. Nasal swabs from someof the greyhounds were also submitted to the Department of DiagnosticMedicine/Pathobiology at the Kansas State University College ofVeterinary Medicine for bacterial culture. Of 70 clinically diseaseddogs tested, Bordetella bronchiseptica was isolated from the nasalcavity of 1 dog, while Mycoplasma spp. were recovered from the nasalcavity of 33 dogs. Pasteurella multocida was commonly recovered from thenasal cavity of dogs with purulent nasal discharges. Two of the dogsthat died in the January 2004 outbreak had scant growth of Escherichiacoli in the lungs postmortem, one dog had scant growth of E. coli andStreptococcus canis, and another had scant growth of Pseudomonasaeruginosa and a yeast. Neither Bordetella bronchiseptica nor Mycoplasmawas isolated from the trachea or lungs of dogs that died.

Virus Isolation from Postmortem Tissues.

Frozen tissues were thawed and homogenized in 10 volumes of minimumessential medium (MEM) supplemented with 0.5% bovine serum albumin (BSA)and antibiotics. Solid debris was removed by centrifugation andsupernatants were inoculated onto cultured cells or into 10-day oldembryonated chicken eggs. Tissue homogenates from greyhounds that diedwere inoculated into diverse cell cultures that supported thereplication of a broad range of viral pathogens. The cell culturesincluded Vero (African green monkey kidney epithelial cells, ATCC No.CCL-81), A-72 (canine tumor fibroblasts, CRL-1542), HRT-18 (human rectalepithelial cells, CRL-11663), MDCK (canine kidney epithelial cells,CCL-34), primary canine kidney epithelial cells (AHDL, CornellUniversity), primary canine lung epithelial cells (AHDL), and primarybovine testicular cells (AHDL). MDCK and HRT cells were cultured in MEMsupplemented with 2.5 ug/mL TPCK-treated trypsin (Sigma); the remainingcell lines were cultured in MEM supplemented with 10% fetal calf serumand antibiotics. Cells were grown in 25 cm² flasks at 37° C. in ahumidified atmosphere containing 5% CO₂. A control culture wasinoculated with the supplemented MEM. The cultures were observed dailyfor morphologic changes and harvested at 5 days post inoculation. Theharvested fluids and cells were clarified by centrifugation andinoculated onto fresh cells as described for the initial inoculation;two blind passages were performed. Hemagglutination activity in theclarified supernatants was determined using chicken or turkey red bloodcells as described (Burleson et al., 1992; Kendal et al., 1982). Forvirus isolation in chicken embryos, 0.1 mL of tissue homogenate wasinoculated into the allantoic sac and incubated for 48 hours at 35° C.After two blind passages, the hemagglutination activity in the allantoicfluids was determined as described (Burleson et al., 1992; Kendal etal., 1982).

RT-PCR, Nucleotide Sequencing, and Phylogenetic Analyses.

Total RNA was extracted from tissue culture supernatant or allantoicfluid using the RNeasy kit (Qiagen, Valencia, Calif.) according tomanufacturer's instructions. The total RNA (10 ng) was reversetranscribed to cDNA using a one-step RT-PCR Kit (Qiagen, Valencia,Calif.) according to manufacturer's instructions. PCR amplification ofthe coding region of the 8 influenza viral genes in the cDNA wasperformed as previously described (Klimov et al., 1992a), usinguniversal gene-specific primer sets. The resulting DNA amplicons wereused as templates for automated sequencing on an Applied Biosystems 3100automated DNA sequencer using cycle sequencing dye terminator chemistry(ABI). Nucleotide sequences were analyzed using the GCG Package©,Version 10.0 (Accelyrs) (Womble, 2000). The Phylogeny Inference Package©Version 3.5 was used to estimate phylogenies and calculate bootstrapvalues from the nucleotide sequences (Felsenstein, 1989). Phylogenetictrees were compared to those generated by neighbor joining analysis withthe Tamura-Nei gamma model implemented in the MEGA© program (Kumar etal., 2004) and confirmed by the PAUP© 4.0 Beta program (SinauerAssociates).

Experimental Inoculation of Dogs.

Four 6-month old specific pathogen-free beagles [(2 males and 2 females(Liberty Research)] were used. Physical examination and baseline bloodtests including complete blood cell count/differential, serum chemistrypanel, and urinalysis determined that the animals were healthy. Theywere housed together in a BSL 2-enhanced facility accredited by theAssociation for Assessment and Accreditation of Laboratory Animal Care.Baseline rectal temperatures were recorded twice daily for 7 days. Thedogs were anesthetized by intravenous injection of propofol (Diprivan®,Zeneca Pharmaceuticals, 0.4 mg/kg body weight to effect) for intubationwith endotracheal tubes. Each dog was inoculated with a total dose of10^(6.6) median tissue culture infectious doses (TCID₅₀) ofA/Canine/Florida/43/2004 (Canine/FL/04) (H3N8) virus with half the doseadministered into the distal trachea through the endotracheal tube andthe other half administered into the deep nasal passage through acatheter. Physical examinations and rectal temperature recordings wereperformed twice daily for 14 days post inoculation (p.i.). Blood samples(4 mL) were collected by jugular venipuncture on days 0, 3, 5, 7, 10,and 14 p.i. Nasal and oropharyngeal specimens were collected withpolyester swabs (Fisher Scientific) from each dog on days 0 to 5, 7, 10,and 14 p.i. The swabs were placed in viral transport medium (Remel) andstored at −80° C. Two dogs (1 male and 1 female) were euthanatized byintravenous inoculation of Beuthanasia-D® solution (1 mL/5 kg bodyweight; Schering-Plough Animal Health Corp) on day 5 p.i. and theremaining 2 dogs on day 14 for postmortem examination. Tissues forhistological analysis were processed as described. Tissues for virusculture were stored at −80° C. This study was approved by the Universityof Florida Institutional Animal Care and Use Committee.

Virus Shedding from Experimentally Inoculated Dogs.

Serial dilutions of lung homogenates and swab extracts, prepared byclarification of the swab transport media by centrifugation, were set upin MEM supplemented with 0.5% BSA and antibiotics. Plaque assays wereperformed as described (Burleson et al., 1992) using monolayers of MDCKcells in 6-well tissue culture plates. Inoculated cell monolayers wereoverlaid with supplemented MEM containing 0.8% agarose and 1.5 ug/mL ofTPCK-trypsin. Cells were cultured for 72 hours at 37° C. in a humidifiedatmosphere containing 5% CO₂ prior to fixation and staining with crystalviolet. Virus concentration was expressed as plaque forming units (PFU)per gram of tissue or per swab.

Immunohistochemistry.

Deparaffinized and rehydrated 5-μm lung tissue sections from thegreyhounds and beagles were mounted on Bond-Rite™ slides (Richard-AllanScientific, Kalamazoo, Mich.) and subsequently treated with proteinase K(DakoCytomation, Carpenteria, Calif.) followed by peroxidase blockingreagent (Dako® EnVision™ Peroxidase Kit, Dako Corp.). The sections wereincubated with 1:500 dilutions of monoclonal antibodies to caninedistemper virus (VMRD, Inc.), canine adenovirus type 2 (VMRD, Inc.),canine parainfluenza virus (VMRD, Inc.), or influenza A H3 (ChemiconInternational, Inc.) for 2 hours at room temperature. Controls includedincubation of the same sections with mouse IgG (1 mg/mL, Serotec, Inc.),and incubation of the monoclonal antibodies with normal canine lungsections. Following treatment with the primary antibodies, the sectionswere incubated with secondary immunoperoxidase and peroxidase substratereagents (Dako® EnVision™ Peroxidase Kit, Dako Corp.) according to themanufacturer's instructions. The sections were counterstained withhematoxylin, treated with Clarifier #2 and Bluing Reagent (Richard-AllanScientific, Kalamazoo, Mich.), dehydrated, and coverslips applied withPermount (ProSciTech).

Hemagglutination Inhibition (HI) Assay.

Serum samples were incubated with receptor destroying enzyme (RDE,Denka) (1 part serum: 3 parts RDE) for 16 hours at 37° C. prior to heatinactivation for 60 minutes at 56° C. Influenza A/Canine/FL/04 (H3N8)virus was grown in MDCK cells for 36-48 hr at 37° C. Virus culturesupernatants were harvested, clarified by centrifugation, and stored at−80° C. The HI assay was performed as described previously (Kendal etal., 1982). Briefly, 4 hemagglutinating units of virus in 25 μl wereadded to an equal volume of serially diluted serum in microtiter wellsand incubated at room temperature for 30 minutes. An equal volume of0.5% v/v turkey erythrocytes was added and the hemagglutination titerswere estimated visually after 30 minutes. The endpoint HI titer wasdefined as the last dilution of serum that completely inhibitedhemagglutination. Seroconversion was defined as ≧4-fold increase in HItiter between paired acute and convalescent samples. Seropositivity of asingle sample was defined as a HI antibody titer ≧1:32.

Microneutralization (MN) Assay.

Neutralizing serum antibody responses to A/Canine/FL/04 (H3N8) weredetected by a MN assay as described previously (Rowe et al., 1999)except that canine sera were RDE-treated as described above prior to theassay. The endpoint titer was defined as the highest dilution of serumthat gave 50% neutralization of 100 TCID₅₀ of virus. Seroconversion wasdefined as ≧4-fold increase in MN titer between paired acute andconvalescent samples. Seropositivity of a single sample was defined as aMN titer ≧1:80.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1

In January 2004, an outbreak of respiratory disease occurred in 22racing greyhounds housed in 2 kennels at a Florida track and the localfarm that supplied dogs to these kennels. There were approximately 60dogs in each kennel building and 300 dogs at the farm. The outbreakoccurred over a 6-day period after which no new cases were identified.Fourteen of the 22 dogs had fevers of 39.5 to 41.5° C., a soft, gaggingcough for 10 to 14 days, and eventual recovery. Of the remaining 8 dogs,6 apparently healthy dogs died unexpectedly with hemorrhage from themouth and nose. Two other dogs were euthanatized within 24 hours ofonset of hemorrhage from the mouth and nose due to rapid deterioration.Both of these dogs had fevers of 41° C. Four of the 8 deaths occurred inthe kennel buildings and 4 occurred at the farm. Fifty percent of thedeaths occurred on day 3 of the outbreak. The 22 dogs ranged in age from17 months to 4 years, but 73% were 17 to 33 months old.

Two clinical syndromes were evident: a milder illness characterized byinitial fever and then cough for 10-14 days (14 dogs) with subsequentrecovery, or a peracute death associated with hemorrhage in therespiratory tract (8 dogs for a mortality rate of 36%). Postmortemexaminations were performed on 6 of the 8 fatal cases. All dogs hadextensive hemorrhage in the lungs, mediastinum, and pleural cavity.Histological examination of the respiratory tract revealed that inaddition to pulmonary hemorrhage, all dogs had tracheitis, bronchitis,bronchiolitis, and suppurative bronchopneumonia (FIGS. 3A and 3B). Theepithelial lining and airway lumens in these tissues were infiltrated byneutrophils and macrophages. Lung homogenates prepared from these dogswere inoculated into a variety of monkey, human, bovine, and canine celllines for virus culture. The lung homogenate from one dog causedcytopathic effects in Madin-Darby canine kidney epithelial cells (MDCK)cultured in the presence of trypsin, and the cell culture supernatantagglutinated chicken red blood cells. Preliminary evidence of aninfluenza type A virus was provided by a commercial ELISA for detectionof the nucleoprotein of influenza A and B viruses, and by PCR analysisusing primers specific for the matrix gene of influenza A viruses. Inaddition, the hemagglutinating activity was inhibited by referenceantisera to the equine influenza A H3 subtype, but not by antiseraspecific for human influenza A subtypes H1-H11 and H13 (Table 3). Tocharacterize the molecular properties of the virus, we determined thenucleotide sequences of the 8 RNA segments of the viral genome. Sequencecomparisons with known influenza virus genes and phylogenetic analysesindicated that the 8 genes of the canine isolate were most similar tothose from contemporary equine influenza A (H3N8) viruses, with whichthey shared 96-97% sequence identity (FIG. 1A, Table 4). In contrast,representative genes from avian, swine, and human influenza A isolateshad <94% identity with the canine isolate (Table 4). These dataidentified the canine isolate A/Canine/Florida/43/2004 (Canine/FL/04) asan influenza A H3N8 virus closely related to contemporary lineages ofequine influenza viruses. Since all genes of the canine isolate were ofequine influenza virus origin, we concluded that the entire genome of anequine influenza virus had been transmitted to the dog.

Example 2

To investigate the role of the Canine/FL/04 virus in the clinical andpathological observations in the greyhounds, we performedimmunohistochemical staining (IHC) on lung tissues using a monoclonalantibody to influenza A H3. Viral H3 antigen was consistently detectedin the cytoplasm of bronchial and bronchiolar epithelial cells,bronchial gland epithelial cells, and macrophages in airway lumens andalveolar spaces (FIG. 2A). These data support a diagnosis of pulmonaryinfection with influenza virus of the H3 subtype in multiple dogs.

Example 3

To determine involvement of a Canine/FL/04-like virus in the etiology ofthe respiratory disease outbreak, we analyzed paired acute andconvalescent sera from 11 sick dogs and 16 asymptomatic contacts byhemagglutination inhibition (HI) and microneutralization (MN).Seroconversion, defined as a ≧4-fold rise in antibody titer toCanine/FL/04 from the acute to convalescent phase, occurred in 8 of 11(73%) sick dogs in both assays (Table 1). Seroconversion occurred in 6of 16 (38%) asymptomatic contacts in the HI assay, while 8 of 16 (50%)seroconverted in the MN assay (Table 1). The seroconversion datademonstrated infection of the dogs with a Canine/FL/04-like virus whichcoincided temporally with the onset of respiratory disease in mostanimals.

Single serum samples were collected 3 months after the outbreak from anadditional 46 asymptomatic dogs housed with the sick dogs. Of these, 43(93%) were seropositive in both assays. For the total population of 73dogs tested, 93% were seropositive in both assays, including 82% (9/11)of the sick dogs and 95% (59/62) of the healthy contacts. The highseroprevalence in dogs with no history of respiratory disease indicatesthat most infections with canine influenza virus are subclinical andsuggest efficient spread of the virus among dogs. It is not known ifsubclinical infections contribute to the spread of the virus.

Example 4

To better understand the capacity of the Canine/FL/04 virus to infectdogs, four 6-month old purpose-bred beagles were each inoculated with10^(6.6) median tissue culture infectious doses (TCID₅₀) by theintratracheal and intranasal routes. All dogs developed a fever (rectaltemperature ≧39° C.) for the first 2 days postinoculation (p.i.), butnone exhibited respiratory symptoms such as cough or nasal dischargeover a 14 day observation period. Virus shedding was examined byquantification of virus in nasal and oropharyngeal swabs. Only 2 of the4 dogs shed detectable amounts of virus. One dog shed virus on days 1and 2 p.i. (1.0-2.5 log₁₀ PFU per swab), whereas the other dog shedvirus for 4 consecutive days after inoculation (1.4-4.5 log₁₀ PFU perswab). Postmortem examination of 2 dogs on day 5 p.i. revealednecrotizing and hyperplastic tracheitis, bronchitis, and bronchiolitissimilar to that found in the spontaneous disease in greyhounds, butthere was no pulmonary hemorrhage or bronchopneumonia. Viral H3 antigenwas detected in the cytoplasm of epithelial cells of bronchi,bronchioles, and bronchial glands by IHC (FIG. 2B). Infectious virus wasrecovered from the lung tissue of one of the dogs. Postmortemexamination of the remaining 2 dogs on day 14 p.i. showed minimalhistological changes in respiratory tissues, no viral H3 antigen by IHC,and no recovery of virus from lung homogenates. Seroconversion in theselatter 2 dogs was detected in MN assays by day 7 p.i., with a further 2-to 3-fold increase in antibody titers by day 14. These resultsestablished the susceptibility of dogs to infection with Canine/FL/04,as evidenced by the febrile response, presence of viral antigen andinfectious virus in the lung parenchyma, histopathological findingstypical for influenza, and seroconversion. The failure to reproducesevere disease and death in the experimentally inoculated beagles is notsurprising since a large proportion of the naturally infected greyhoundswere asymptomatic.

Example 5

To investigate whether a Canine/FL/04-like influenza virus hadcirculated among greyhound populations in Florida prior to the January2004 outbreak, archival sera from 65 racing greyhounds were tested forthe presence of antibodies to Canine/FL/04 using the HI and MN assays.There were no detectable antibodies in 33 dogs sampled from 1996 to1999. Of 32 dogs sampled between 2000 and 2003, 9 were seropositive inboth assays—1 in 2000, 2 in 2002, and 6 in 2003 (Table 5). Theseropositive dogs were located at Florida tracks involved in outbreaksof respiratory disease of unknown etiology from 1999 to 2003, suggestingthat a Canine/FL/04-like virus may have been the causative agent ofthose outbreaks. To investigate this possibility further, we examinedarchival tissues from greyhounds that died from hemorrhagicbronchopneumonia in March 2003. Lung homogenates inoculated into MDCKcells and chicken embryos from one dog yielded H3N8 influenza virus,termed A/Canine/Florida/242/2003 (Canine/FL/03). Sequence analysis ofthe complete genome of Canine/FL/03 revealed >99% identity toCanine/FL/04 (Table 4), indicating that Canine/FL/04-like viruses hadinfected greyhounds prior to 2004.

Example 6

From June to August 2004, respiratory disease outbreaks occurred inthousands of racing greyhounds at 14 tracks in Florida, Texas, Alabama,Arkansas, West Virginia, and Kansas.

Officials at some of these tracks estimated that at least 80% of theirdog population had clinical disease. Most of the dogs had clinical signsof fever (≧39° C.) and cough similar to the dogs in the January 2004outbreak, but many dogs also had a mucopurulent nasal discharge.Multiple deaths were reported but an accurate mortality rate could notbe determined.

We collected paired acute and convalescent sera from 94 dogs located at4 Florida tracks: 56% of these dogs had ≧4-fold rises in antibody titersto Canine/FL/04, and 100% were seropositive (Table 6). Convalescent serafrom 29 dogs in West Virginia and Kansas also had antibodies toCanine/FL/04. We isolated influenza A (H3N8) virus from the lungs of agreyhound that died of hemorrhagic bronchopneumonia at a track in Texas.Sequence analysis of the entire genome of this isolate, namedA/Canine/Texas/1/2004 (Canine/TX/04), revealed 99% identity toCanine/FL/04 (Table 4). The isolation of three closely related influenzaviruses from fatal canine cases over a 13-month period and fromdifferent geographic locations, together with the substantialserological evidence of widespread infection among racing greyhounds,suggested sustained circulation of a Canine/FL/04-like virus in the dogpopulation.

Phylogenetic analysis of the HA genes of Canine/FL/03, Canine/FL/04, andCanine/TX/04 showed that they constitute a monophyletic group withrobust bootstrap support that was clearly distinct from contemporary H3genes of equine viruses isolated in 2002 and 2003 (FIG. 1B). Phylogenticanalysis and pairwise nucleotide sequence comparisons of the other 7genomic segments supported the segregation of the canine genes as adistinct sub-lineage most closely related to the equine virus lineage(data not shown, and Table 4). The clustering of the canine influenzagenes as a monophyletic group separate from equine influenza is alsosupported by the presence of 4 signature amino acid changes in the HA(Table 2). Together with the serological results from 2003 and 2004,these data are consistent with a single virus transmission event fromhorses to dogs with subsequent horizontal spread of the virus in thegreyhound population. However, repeated introductions of this uniquelineage of influenza virus from an unidentified reservoir species cannot be formally excluded, unlikely as it may be.

The viral HA is a critical determinant of host species specificity ofinfluenza virus (Suzuki et al., 2000). To identify residues within HAthat may be associated with adaptation to the canine host, we comparedthe deduced amino acid sequence of canine HAs to those of contemporaryequine viruses. Four amino acid changes differentiate the equine andcanine mature HA consensus amino acid sequences: N83S, W222L, I328T, andN483T (see Table 2). The canine viruses have an amino acid deletion whencompared to the consensus equine sequences. Therefore, amino acidposition 7 in the HA equine sequence is position 6 in the HA caninesequence, amino acid position 29 in the HA equine sequence is position28 in the HA canine sequence, amino acid position 83 in the HA equinesequence is position 82 in the HA canine sequence, etc. Thus, the foursubstituted amino acids are at position 82, 221, 327, and 482 of theamino acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 34. Thesubstitution of serine for asparagine at consensus sequence position 83is a change of unknown functional significance since various polarresidues are found in H3 molecules from other species. The strictlyconserved isoleucine at consensus sequence position 328 near thecleavage site of the H3 HA has been replaced by threonine. The pivotalrole of HA cleavage by host proteases in pathogenesis suggests that thischange merits further study. The substitution of leucine for tryptophanat consensus sequence position 222 is quite remarkable because itrepresents a non-conservative change adjacent to the sialic acid bindingpocket which could modulate receptor function (Weis et al., 1988).Interestingly, leucine at position 222 is not unique to canine H3 HAsince it is typically found in the H4, H8, H9, and H12 HA subtypes(Nobusawa et al., 1991; Kovacova et al., 2002). The leucine substitutionmay be more compatible with virus specificity for mammalian hosts sinceinfections of swine with subtype H4 (Karasin et al., 2000) and humansand swine with subtype H9 (Peiris et al., 1999) viruses have beenreported. The substitution of asparagine with threonine at consensussequence position 483 resulted in the loss of a glycosylation site inthe HA2 subunit that is conserved in all HA subtypes (Wagner et al.,2002). Although the importance of these amino acid changes in the HA foradaptation of an equine virus to dogs remains to be determined, similaramino acid changes have been observed previously in association withinterspecies transfer (Vines et al., 1998; Matrosovich et al., 2000).Amino acid differences between other influenza viral proteins of theinvention and equine consensus sequence are shown in Tables 19 to 25.

The source of the equine influenza virus that initially infected racinggreyhounds remains speculative. Kennels at greyhound racetracks are notlocated near horses or horse racetracks, suggesting that contact betweengreyhounds and shedding horses is not a sufficient explanation for themultiple outbreaks in different states in 2004. A potential source ofexposure to the equine virus is the feeding of horsemeat to greyhounds,whose diet is supplemented with raw meat supplied by packing houses thatrender carcasses, including horses which could carry influenza.Precedents for this mode of infection include reports of interspeciestransmission of H5N1 avian influenza virus to pigs and zoo felids fedinfected chicken carcasses (Webster, 1998; Keawcharoen et al., 2004;Kuiken et al., 2004). Although this is a plausible route for the initialintroduction of equine influenza into dogs, it does not explain therecent multiple influenza outbreaks in thousands of dogs in differentstates. Our experimental inoculation study demonstrated the presence ofvirus in the nasal passages and oropharynx of dogs, albeit at modesttiters. Nevertheless, these results indicate that shedding is possible,and that dog-to-dog transmission of virus by large droplet aerosols,fomites, or direct mucosal contact could play a role in theepizootiology of the disease.

The interspecies transfer of a whole mammalian influenza virus to anunrelated mammal species is a rare event. Previous studies have providedlimited serological or virological evidence, but not both, of transientinfection of dogs with human influenza A (H3N2) viruses (Nikitin et al.,1972, Kilbourne, et al., 1975; Chang et al., 1976; Houser et al., 1980).However, there was no evidence of sustained circulation in the caninehost. Although direct transfer of swine influenza viruses from pigs topeople is well-documented (Dacso et al., 1984; Kimura et al., 1998;Patriarca et al., 1984; Top et al., 1977), there is no evidence foradaptation of the swine viruses in human hosts. In this report, weprovide virological, serological, and molecular evidence forinterspecies transmission of an entire equine influenza A (H3N8) virusto another mammalian species, the dog. Unique amino acid substitutionsin the canine virus HA, coupled with serological confirmation ofinfection of dogs in multiple states in the U.S., suggest adaptation ofthe virus to the canine host. Since dogs are a primary companion animalfor humans, these findings have implications for public health; dogs mayprovide a new source for transmission of novel influenza A viruses tohumans.

TABLE 1 Antibody response to A/Canine/Florida/43/2004 (H3N8). Sick Dogs(11)^(a) Healthy Contacts (16)^(b) Response HI^(c) SN^(d) HI SNSeroconversion (%)^(e) 73 73 38 50 Seropositive (%)^(f) 82 82 100 100Geometric mean titer^(g) 329 424 268 431 ^(a)Number of dogs withclinical signs of disease. ^(b)Number of asymptomatic dogs housed incontact with clinically diseased dogs. ^(c)Hemagglutination-inhibition(HI) assay using A/Canine/Florida/43/2004 virus. ^(d)Microneutralization(MN) assay using A/Canine/Florida/43/2004 virus. ^(e)Percentage of dogswith at least a 4-fold increase in antibody titer in paired acute andconvalescent sera. ^(f)Percentage of dogs with a positive antibody titer(HI titer ≧32: MN titer ≧80) in the convalescent sera. ^(g)Geometricmean antibody titer for the convalescent sera.

TABLE 2 Amino acid differences between the canine and equine H3hemagglutinins. Equine H3 Potential functional consensus Can/FL/03Can/FL/04 Can/TX/04 significance G7* D — † — D also found in duck andhuman H3 HA I29 — M M I is conserved in H3 HAs from all species N83 S SS Various polar amino acids present at this position in H3 HAs of otherspecies S92 — N — N is present in some duck H3 HAs L118 — — V L isconserved in all H3 HAs W222 L L L W is conserved in most H3 HAs of allspecies; located near the receptor binding site A272 V A V V is presentin some recent equine isolates I328 T T T T is strictly conserved in allavian, swine or humans H3 HAs N483 T T T N occurs in all H3 and other HAsubtypes. Replacement results in loss of a glycosylation site. K541 — R— Basic amino acid conservative change *Amino acid residue (singleletter code) and position in the mature H3 HA. The amino acid code is: A= alanine, D = aspartic acid, G = glycine, I = isoleucine, K = lysine, L= leucine, M = methionine, N = asparagine, R = arginine, S = serine, T =threonine, V = valine, W = tryptophan. † Denotes no change from theconsensus equine H3 HAs.

TABLE 3 Hemagglutination inhibition of a virus isolate by referenceantisera to different HA subtypes. Reference Antisera HA Specificity HItiter^(a) Puerto Rico/8/34 H1 5 Swine/Iowa 15/30 H1 5 Singapore/01/57 H25 Shanghai/11/87 H3^(b) 5 Equine/Miami/1/63 H3 160Duck/Czechoslovakia/56 H4 5 Tern/South Africa/61 H5 5Turkey/Massachussetts/65 H6 5 Fowl Plague/Dutch/27 H7 5 FowlPlague/Rostock/34 H7 5 Equine/Prague/1/56 H7 5 Turkey/Ontario/6118/68 H85 Quail/Hong Kong/G1/97 H9^(b) 5 Chicken/Hong Kong/G9/97 H9^(b) 5Chicken/Germany/49 H10 5 Duck/England/56 H11 5 Gull/Maryland/704/77 H135 Normal sheep serum — 5 Normal ferret serum — 5 ^(a)Hemagglutinationinhibition titer to virus isolate from dog # 43. ^(b)Polyclonal antiserawere produced in ferrets, whereas all other antisera were produced insheep or goats.

TABLE 4 Sequence homology of A/Canine/Florida/43/2004 (H3N8) genes toequine, avian, swine, and human strains of influenza A. Gene EquineAvian Swine Human PB2 96.9 (98.7)^(a) 88.6 (96.8) 87.9 (96.8) 86.2(96.4) DQ124147 Eq/Kentucky/2/8 Mall/Alberta/98/85 Sw/Ontario/ PR/8/34M73526 AY633315 01911-1/99 (HK/213/03) AF285892 AF389115 (AY576381) PB197.1 (98.8) 83.9 (97.1) 83.9 (97.1) 83.9 (97.1) DQ124148Eq/Tennessee/5/86 Ck/BritishColumbia/04 Sw/Korea/S109/04 WSN/33 M25929(Gull/Md/704/77) (Sw/Saskatch/ (Sing/1/57) AY61675 (M25933) 18789/02)J02178 AY790287 (M25924) (AY619955) PA 96.3 (97.5) 87.0 (94.3) 84.3(94.6) 83.8 (93.4) DQ124149 M26082 Ck/Chile/4591/02 Sw/Hong Kong/Taiwan/2/70 Eq/Tennesee/5/86 (Ostrich/SA/08103/95) 126/02 (Viet Nam/AY303660(AF508662) M26081 1203/04) AY210199 (AY818132) HA (H3) 97.4(97.1) 80.7 (89.0) 80.0 (87.7) 81.8 (87.9) DQ124190 Eq/FL/1/93Dk/Norway/1/03 Sw/Ontario/42729a/01 HK/1/68 L39916 AJ841293 AY619977AF348176 NP 96.6 (97.9) 87.9(95.1) 85.4 (93.5) 84.7 (93.0) DQ124150Eq/Tennesee/5/86 Ck/Chile/176822/02 Sw/Ontario/42729a/01 HK/1073/99M30758 AY303658 (Sw/Fujian/1/2003) (Hong Kong/ AY619974 538/97)(AY747611) AF255742 (AF255751) NA (N8) 96.8 (97.0) 84.0 (85.2) na^(b)na^(b) DQ124151 Eq/Tennesee/5/86 Dk/NJ/2000 L06583 L06583 M 97.9 (95.7)94.1 (94.0) 93.7 (93.5) 91.2 (95.4) DQ124152 Eq/Tennesee/5/86Tky/Mn/833/80 Sw/Saskatchewan/ WSN/33 (Eq/Kentucky/92) AF001683 18789/02(Hong Kong/ M63529 M63527 1073/99) (AF001683) J02177 (AJ278646) NS 97.5(95.7) 92.0 (90.4) 91.1 (89.1) 91.4 (90.0) DQ124153 Eq/Tn/5/86Mal/NY/6750/78 Sw/China/8/78 Brevig (Eq/Kentucky/92) M80945(Sw/Korea/s452/04) Mission/1/18 M80973 M80968 (AY790309) AF333238(AF001671) ^(a)Percent nucleotide and amino acid (in parentheses)sequence identity of A/Canine/Florida/43/2004 (H3N8) genes to the mosthomologous gene of influenza virus virus isolates from the species,followed by their Genbank sequence database accession numbers. ^(b)Notapplicable: N8 neuraminidase was never reported in human or swineviruses.

TABLE 5 Antibody titers to A/canine/Florida/43/2004 (H3N8) in greyhoundserum collected from 1996 to 2003. Year^(a) 1996 1997 1998 2000 20022003 No. of dogs tested 8 6 19 4 6 22 No. of seropositive 0 0 0 1 2 6dogs Antibody titers^(b) 512 232, 524 280-2242 ^(a)The year of serumsample collection from racing greyhounds in Florida.^(b)Microneutralization assay antibody titers for seropositive dogs,including the range for the six 2003 seropositive dogs.

TABLE 6 Antibody response to A/canine/Florida/43/2004 (H3N8) in racinggreyhounds at 4 Florida tracks in June 2004. Response Track A Track BTrack C Track D Number of dogs tested^(a) 37 10 22 25 Seroconversion(%)^(b) 46 90 100 64 Seropositive (%)^(c) 100 100 100 100 Geometric meantiter^(d) 401 512 290 446 ^(a)Number of clinically diseased dogs testedby HI using A/canine/Florida/43/2004 (H3N8). ^(b)Percentage of dogs with≧4-fold increase in antibody titer between acute and convalescent sera.^(c)Percentage of dogs with a positive antibody titer (HI titer > 16) inthe convalescent sera. ^(d)Geometric mean antibody titer for theconvalescent sera.

Materials and Methods for Examples 7-11 Canine Tissues

Postmortem examinations were performed by the Anatomic Pathology Serviceat the University of Florida College of Veterinary Medicine on 6 mixedbreed dogs that died in April/May 2005 during an influenza outbreak in ashelter facility in northeast Florida, and on a pet Yorkshire Terrierdog that died in May 2005 during an influenza outbreak in a veterinaryclinic in southeast Florida. Tissues were fixed in 10% neutral bufferedformalin, embedded in paraffin, and 5-μm sections were stained withhematoxylin and eosin for histopathologic diagnosis. Unfixed tissueswere stored at −80° C. pending virological analyses.

RNA Extraction from Canine Tissue Samples

Frozen lung tissues from each of the 7 dogs were thawed and homogenizedin minimum essential medium (MEM) supplemented with 0.5% bovine serumalbumin (BSA) and antibiotics (gentamycin and ciprofloxacin) using adisposable tissue grinder (Kendall, Lifeline Medical Inc., Danbury,Conn.). Total RNA was extracted using a commercial kit (RNeasy® MiniKit, QIAGEN Inc., Valencia, Calif.) according to manufacturer'sinstructions and eluted in a final volume of 60 μL of buffer. Total RNAwas also extracted from lung tissue collected from dogs withoutrespiratory disease.

Real-Time RT-PCR

A single-step quantitative real-time RT-PCR was performed on total RNAextracted from the canine tissue samples using the QuantiTect® ProbeRT-PCR Kit containing ROX as a passive reference dye (QIAGEN Inc.,Valencia, Calif.). Briefly, 2 primer-probe sets were used for detectionof influenza A sequences in each sample (Table 7). One primer-probe setwas selective for canine hemagglutinin (H3) gene sequences. The otherprimer-probe set targeted a highly conserved region of the matrix (M)gene of type A influenza virus. For each real-time RT-PCR reaction, 5 μLof extracted total RNA were added to a reaction mixture containing 12.5μL of 2X QuantiTech® Probe RT-PCR Master Mix, 0.25 μL of QuantiTech® RTMix, forward and reverse primers (0.4 μM final concentration for each),probe (0.1 μM final concentration) and RNase-free water in a finalvolume of 25 μL. The TaqMan® Ribosomal RNA Control Reagents (AppliedBiosystems, Foster City, Calif.) were used according to manufacturer'sinstructions for detection of 18S rRNA as an endogenous internal controlfor the presence of RNA extracted from the canine tissue samples.

Quantitative one-step real-time RT-PCR was performed on the reactionmixtures in a Mx3000P® QPCR System (Stratagene, La Jolla, Calif.).Cycling conditions included a reverse transcription step at 50° C. for30 minutes, an initial denaturation step at 95° C. for 15 minutes toactivate the HotStarTaq® DNA polymerase, and amplification for 40cycles. Each amplification cycle included denaturation at 94° C. for 15seconds followed by annealing/extension at 60° C. for 1 minute. The FAM(emission wavelength 518 nm) and VIC (emission wavelength 554 nm)fluorescent signals were recorded at the end of each cycle. Thethreshold cycle (Ct) was determined by setting the thresholdfluorescence (dR) at 1000 in each individual experiment. The Mx3000P®version 2.0 software program (Stratagene, La Jolla, Calif.) was used fordata acquisition and analysis. Samples were considered positive forinfluenza A virus when the threshold cycle (Ct) for the H3 or M gene was3 units smaller than the Ct for lung tissues from dogs withoutrespiratory disease. The positive control consisted of amplification ofRNA extracted from A/canine/FL/242/03 (H3N8) virus.

Virus Isolation in MDCK Cells

Frozen lung tissues from each of the 7 dogs were thawed and homogenizedin 10 volumes of Dulbecco's Modified Eagle Medium (DMEM) supplementedwith 0.5% (BSA) and antibiotics (gentamycin and ciprofloxacin). Soliddebris was removed by centrifugation and supernatants were inoculatedonto Madin-Darby canine kidney (MDCK) cells cultured in DMEMsupplemented with 1 μg/mL TPCK-treated trypsin (Sigma-Aldrich Corp., St.Louis, Mo.) and antibiotics (gentamycin and ciprofloxacin). Cells weregrown in 25 cm² flasks at 37° C. in a humidified atmosphere containing5% CO₂. The cultures were observed daily for morphologic changes andharvested at 5 days post inoculation. The harvested cultures wereclarified by centrifugation and the supernatants inoculated onto freshMDCK cells as described for the initial inoculation; two additionalpassages were performed for samples that did not show evidence ofinfluenza virus by hemagglutination or RT-PCR. Hemagglutination activityin the clarified supernatants was determined using 0.5% turkey red bloodcells as previously described (Burleson, F. et al., 1992; Kendal, P. etal., 1982). RT-PCR was performed as described below.

Virus Isolation in Embryonated Chicken Eggs

Homogenates were prepared from frozen lung tissues as described abovefor inoculation of MDCK cells. The homogenates (0.2 mL) were inoculatedinto the allantoic sac of 10-day old embryonated chicken eggs. After 48hours of incubation at 35° C., the eggs were chilled at 4° C. overnightbefore harvesting the allantoic fluid. Hemagglutination activity in theclarified supernatants was determined using 0.5% turkey red blood cellsas previously described (Burleson, F. et al., 1992; Kendal, P. et al.,1982). RT-PCR was performed as described below. Two additional passagesin embryonated eggs were performed for samples that did not showevidence of influenza virus after the initial inoculation.

RT-PCR, Nucleotide Sequencing, and Phylogenetic Analyses

Viral RNA was extracted from MDCK supernatant or allantoic fluid usingthe QIAamp® Viral RNA Mini Kit (QIAGEN Inc., Valencia, Calif.) accordingto manufacturer's instructions. The viral RNA was reverse transcribed tocDNA using the QIAGEN® OneStep RT-PCR Kit (QIAGEN Inc., Valencia,Calif.) according to manufacturer's instructions. PCR amplification ofthe coding region of the 8 influenza viral genes in the cDNA wasperformed as previously described (Klimov, A. et al., 1992b), usinguniversal gene-specific primer sets (primer sequences available onrequest). The resulting DNA amplicons were used as templates forautomated sequencing in the ABI PRISM® 3100 automated DNA sequencerusing cycle sequencing dye terminator chemistry (Applied Biosystems,Foster City, Calif.). Nucleotide sequences were analyzed using theLasergene 6 Package® (DNASTAR, Inc., Madison, Wis.). The PHYLIP Version3.5© software program was used to estimate phylogenies and calculatebootstrap values from the nucleotide sequences (Felsenstein, J., 1989).Phylogenetic trees were compared to those generated by neighbor joininganalysis with the Tamura-Nei model implemented in the MEGA© program(Kumar, S. et al., 2004) and confirmed by the PAUP© 4.0 Beta program(Sinauer Associates, Inc., Sunderland, Mass.).

Hemagglutination Inhibition (HI) Assay

Serum samples were incubated with receptor destroying enzyme (RDE, DENKASEIKEN Co., Ltd., Tokyo, Japan) (1 part serum: 3 parts RDE) for 16 hoursat 37° C. prior to heat inactivation for 30 minutes at 56° C. InfluenzaA/Canine/Jacksonville/05 (H3N8) virus was grown in MDCK cells for 72 hrsat 37° C. in 5% CO₂. Virus culture supernatants were harvested,clarified by centrifugation, and stored at −80° C. All other virusesused in the HI assay were grown in 10-day old embryonated chicken eggsfrom which allantoic fluid was collected and stored at −80° C. The HIassay was performed as described previously (Kendal, P. et al., 1982).Briefly, 4 hemagglutinating units of virus in 25 μl were added to anequal volume of serially diluted serum in 96-well plastic plates andincubated at room temperature for 30 minutes. An equal volume of 0.5%turkey erythrocytes was added and the hemagglutination titers wereestimated visually after 30 minutes. The endpoint HI titer was definedas the last dilution of serum that completely inhibitedhemagglutination.

Example 7 Clinical Cases

In April and May 2005, a previously described (Crawford, P. C. et al.,2005) respiratory disease outbreak occurred in dogs housed in a shelterfacility in northeast Florida. The outbreak involved at least 58 dogsranging in age from 3 months to 9 years, and included purebred dogs aswell as mixed breeds. The most common clinical signs were purulent nasaldischarge and a cough for 7 to 21 days. Of the 43 dogs that had clinicaldisease for ≧7 days, 41 had HI antibody titers to canine/FL/04 (H3N8)ranging from 32 to >1024. At least 10 dogs progressed to pneumonia, ofwhich 6 were euthanized. These 6 mixed breed dogs included 3 males and 3females ranging in age from 4 months to 3 years. The duration ofclinical signs ranged from 2 to 10 days at the time of euthanasia. Onpostmortem examination, these dogs had pulmonary congestion and edema.Histological examination of the respiratory tract revealed rhinitis,tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia.There was epithelial cell necrosis and erosion in the trachea, bronchi,bronchioles, and bronchial glands. The respiratory tissues wereinfiltrated by neutrophils and macrophages.

In May 2005, a respiratory disease outbreak occurred in 40 pet dogs at aveterinary clinic in southeast Florida. The most common clinical signswere purulent nasal discharge and a cough for 10 to 30 days. Of the 40dogs, 17 were seropositive for canine/FL/04 (H3N8) with HI antibodytiers ranging from 32 to >1024. Seroconversion occurred in 10 dogs forwhich paired acute and convalescent sera were available. Three dogsprogressed to pneumonia. One of these dogs, a 9-year old male YorkshireTerrier, died 3 days after onset of clinical signs. This dog hadtracheobronchitis, pulmonary edema and congestion, and severebronchopneumonia. Similar to the 6 shelter dogs, there was epithelialcell necrosis and erosion of the airways and neutrophilic infiltrates inthe tissues.

Example 8 Real-Time RT-PCR and Viral Isolation

Lung tissues from the 7 dogs were analyzed by quantitative real-timeRT-PCR assays that detect the M gene of influenza type A and the H3 geneof canine H3N8 influenza A virus. The lungs from all 7 dogs werepositive for both the influenza A M gene and the canine influenza H3gene (Table 8). After 3 passages in MDCK cells, influenza A subtype H3N8virus was isolated from the lungs of a shelter dog that died after 3days of pneumonia. This virus was named A/canine/Jacksonville/05 (H3N8)(canine/Jax/05). After 2 passages in embryonated chicken eggs, influenzaA subtype H3N8 virus was recovered from the lungs of the pet dog thatalso died after 3 days of pneumonia. This virus was namedA/canine/Miami//05 (H3N8) (canine/Miami/05).

Example 9 Genetic Analyses of the Canine Influenza A H3N8 Isolates

Sequence analyses of canine/Jax/05 and canine/Miami/05 revealed thattheir hemagglutinin (HA) genes were 98% identical to the canine/FL/04,canine/TX/04, and canine/Iowa/05 isolates recovered from the lungs ofracing greyhounds that died of pneumonia during influenza outbreaks attracks in 2004 and 2005 (Crawford, P. C. et al., 2005; Yoon K-Y. et al.,2005). In addition, the HA genes of canine/Jax/05 and canine/Miami/05were 98% identical to contemporary equine influenza viruses isolatedafter the year 2000. Phylogenetic comparisons of the HA genes showedthat the canine/Jax/05 and canine/Miami/05 viruses were clustered withthe canine/FL/04, canine/TX/04, and canine/Iowa/05 greyhound isolatesand contemporary equine isolates, forming a distinct group from theolder equine viruses isolated in the early 1990's (FIG. 4). Furthermore,the canine/Jax/05, canine/Miami/05, and canine/Iowa/05 isolates weremore closely related to canine/Tx/04 than to either canine/FL/04 orcanine/FL/03. The 2005 isolates formed a subgroup that appears to branchoff from the earlier 2003 and 2004 canine viruses with differences atapproximately 10 parsimony-informative sites. These differences supportthe hypothesis that canine influenza virus is being transmittedhorizontally from dog-to-dog as opposed to being reintroducedperiodically from an outside source. The accumulation of mutations from2003 to 2005 illustrates the ongoing process of adaptation that thevirus must undergo after being transmitted to a new host, as is expectedto have happened for the canine influenza viruses.

Example 10 Amino Acid Analyses of the Canine Influenza A H3N8 Isolates

There were conserved amino acid substitutions in all 6 canine isolatesthat differentiated them from contemporary equine influenza viruses(Table 9). These conserved substitutions were I15M, N83S, W222L, I328T,and N483T. Phylogenetic comparisons of the mature HA protein showed thatthe canine/Jax/05, canine/Miami/05, and canine/Iowa/05 viruses formed asubgroup with the canine/TX/04 isolate (FIG. 4). There were 3 amino acidchanges (L118V, K261N, and G479E) that differentiated this subgroup fromthe other canine viruses (Table 9). There were 2 amino acid changes(F79L and G218E) that differentiated the 2005 isolates from theircanine/TX/04 root. Furthermore, the 2005 isolates from non-greyhounddogs, canine/Jax/05 and canine/Miami/05, differed from thecanine/Iowa/05 greyhound isolate by one amino acid change, R492K.Finally, canine/Jax/05 differed from canine/Miami/05 at a single aminoacid, S107P. In all other H3N8 equine and canine viruses, S is conservedat position 107 except for A/Equine/Jilin/1/89 which has a T (Guo Y. etal., 1992).

Example 11 Antigenic Analyses of the Canine Influenza A H3N8 Isolates

Hemagglutination inhibition (HI) tests were performed using an antigenpanel of older and contemporary equine influenza viruses and the canineinfluenza viruses, and serum collected in 2005 from horses and dogs thathad been infected with influenza virus (Table 10). Serum from ferretsimmunized against canine/FL/04 was also included in the analyses. The HIantibody titers in equine serum were 8 to 16-fold higher when testedwith contemporary equine viruses compared to older isolates, butdecreased by at least 4-fold when tested with the canine viruses. Thecanine serum was nonreactive with the older equine viruses, but theantibody titers increased 4-fold when tested with contemporary equineisolates and canine isolates. This was also observed for the serum fromferrets immunized against canine influenza virus. These seroreactivitypatterns demonstrated the antigenic similarity between the canineinfluenza viruses and contemporary equine influenza viruses and wereconsistent with the phylogenetic analyses. The antibody titers inequine, canine, and ferret sera to the canine/Miami/05 isolate weresimilar to those for the 2003 and 2004 canine isolates. However, thetiters were 2 to 4-fold lower for the canine/Jax/05 isolate. Thissuggests that canine/Jax/05 is antigenically distinct from the othercanine isolates, which may in part be related to the single amino acidchange at position 107 in the mature HA.

TABLE 7Primers and probes for quantitative real-time RT-PCR analysis for the matrixgene of influenza A virus and the H3 gene of canine influenza A (H3N8).Primer Target Sequence Application Ca-H3-F387 H3 (nt 387-406)5′-tatgcatcgctccgatccat-3′ Forward primer for H3 (SEQ ID NO: 79)Ca-H3-R487 H3 (nt 487-467) 5′-gctccacttcttccgttttga-3′Reverse primer for H3 (SEQ ID NO: 80) Ca-H3-P430 H3 (nt 430-459)FAM-aattcacagcagagggattcacatggacag-BHQ1 TaqMan ®  probe (SEQ ID NO: 81)FluA-M-F151 M (nt 151-174) 5′-catggartggctaaagacaagacc-3′^(a)Forward primer for M (SEQ ID NO: 82) FluA-M-R276 M (nt 276-253)5′-agggcattttggacaaakcgtcta-3′ Reverse primer for M (SEQ ID NO: 83)FluA-M-P218 M (nt 218-235) FAM-acgcTcaccgTgcccAgt-BHQ1^(b) TaqMan ® probe (SEQ ID NO: 84) ^(a)Underlined letter r represents nucleotide aor g and underlined letter k represents nucleotide g or t. ^(b)Uppercaseletters represent locked nucleic acid residues.

TABLE 8 Quantitative real-time RT-PCR and viral isolation performed onlung tissues from dogs that died from pneumonia during respiratorydisease outbreaks in a shelter and veterinary clinic in Florida.Duration of clinical Real-time RT-PCR Virus Dog ID Location disease M(Ct) HA (Ct) Isolation A/canine/FL/242/03 positive control 28.15 27.361079 Shelter 2 days 29.81 28.84 none (NE FL) 1078 Shelter 3 days 30.3729.71 MDCK (NE FL) 3^(rd) passage 318 Shelter 9 days 33.89 32.97 none(NE FL) 320 Shelter 10 days  39.44 37.09 none (NE FL) 319 Shelter 6 days33.87 32.23 none (NE FL) 1080 Shelter 6 days 38.87 38.23 none (NE FL)374 Veterinary 3 days 24.05 22.65 Egg clinic 2^(nd) passage (SE FL)

TABLE 9 Amino acid comparison of the mature HA for canine influenzaviruses and contemporary equine influenza viruses. Amino Acid 7 15 54 7879 83 92 107 118 159 218 222 261 328 479 483 492 541 A/equine/KY/5/02 GI N V F N S S L N G W K I G N R K A/equine/MA/213/03 . . . A . . . . . S. . . . . . . . A/equine/OH/1/03 D . K A . . . . . S . . . . . . . .A/canine/FL/242/03 . M K A . S . . . S . L . T . T . . A/canine/FL/43/04. M K A . S N . . S . L . T . T . R A/canine/TX/1/04 . M K A . S . . V S. L N T E T . . A/canine/Iowa/05 . M K A L S . . V S E L N T E T . .A/canine/Miami/05 . M K A L S . . V S E L N T E T K .A/canine/Jacksonville/05 . M K A L S . P V S E L N T E T K .

TABLE 10 Antibody titers in equine, canine, and ferret serum to olderand contemporary equine influenza viruses and canine influenza viruses.Serum antibody titers^(a) Antigens Equine Canine Ferret^(b)equine/Miami/63 40 <10 16 equine/Ky/86 40 40 32 equine/KY/92 40 <10 32equine/NY/99 320 40 128 equine/KY/05/02 320 160 256 equine/MA/213/03 640160 512 equine/OH/01/03 640 160 512 canine/FL/03 160 160 512canine/FL/04 160 80 512 canine/Tx/04 160 160 512 canine/Miami/05 160 80256 canine/Jax/05 40 40 128 ^(a)Antibody titers were determined in ahemagglutination inhibition assay performed with serial dilutions ofequine, canine, or ferret serum and the viruses listed in the antigencolumn. ^(b)Serum from ferrets immunized with canine/FL/04 virus.

Materials and Examples Methods for Examples 12-15 Canine Influenza VirusInoculum.

The virus inoculum was prepared by inoculation of Madin-Darby caninekidney (MDCK) epithelial cells with a stock of A/canine/FL/43/04 (H3N8)representing passage 3 of the original isolate previously described(Crawford et al., 2005). The inoculated MDCK cells in Dulbecco's MinimalEssential Media (DMEM) supplemented with 1 μg/mL TPCK-treated trypsin(Sigma-Aldrich Corp., St. Louis, Mo.) and antibiotics (gentamycin andciprofloxacin) were grown in 250 cm² flasks at 37° C. in a humidifiedatmosphere containing 5% CO₂. The cultures were observed daily formorphologic changes and harvested at 5 days post inoculation. Theharvested cultures were clarified by centrifugation and the supernatantswere stored at −80° C. pending inoculation of dogs. An aliquot ofsupernatant was used for determination of virus titer by the Reed andMuench method. The titer was 10⁷ median tissue culture infectious doses(TCID₅₀) of A/canine/Florida/43/2004 (canine/FL/04) per mL.

Experimental Inoculation.

Eight 4-month old colony bred mongrel dogs (Marshall BioResources, NorthRose, N.Y.) (4 males and 4 females) were used for the experimentalinoculation study approved by the University of Florida InstitutionalAnimal Care and Use Committee. The dogs' body weights ranged from 13 to17 kg. The dogs were healthy based on physical examinations, baselineblood tests, and recording of body temperatures for 2 weeks prior toinoculation. All dogs were free from prior exposure to canine influenzavirus based on serology tests performed on paired serum samplescollected at the time of arrival into the facility and 2 weeks later.The dogs were anesthetized by intravenous injection of propofol(Diprivan®, Zeneca Pharmaceuticals, 0.4 mg/kg body weight to effect) forintubation with endotracheal tubes. Six dogs (3 males and 3 females)were each inoculated with 10⁷ TCID₅₀ of canine/FL/04 virus in 5 mL ofsterile saline administered into the distal trachea through a smalldiameter rubber catheter inserted into the endotracheal tube. Two dogs(1 male and 1 female) were sham-inoculated with an equal volume ofsterile saline. The sham-inoculated control dogs were housed in adifferent room from the virus-inoculated dogs and cared for by differentpersonnel. Physical examinations and rectal temperature recordings wereperformed twice daily for 6 days post inoculation (p.i.).

Pharyngeal and Rectal Swab Collection.

To monitor for virus shedding, oropharyngeal specimens were collectedtwice daily from each dog on days 0 to 6 p.i. using polyester swabs(Fisher Scientific International Inc., Pittsburgh, Pa.). The swabs wereplaced in 1 mL of sterile phosphate-buffered saline (PBS) containing0.5% bovine serum albumin (BSA). Rectal swabs were collected from eachdog daily from days 0 to 6. Swab extracts were prepared by clarificationof the swab transport media by centrifugation. An aliquot of swabextract was tested immediately for influenza A virus nucleoprotein usingthe Directigen™ commercial immunoassay kit (BD, Franklin Lakes, N.J.)according to the manufacturer's instructions. The remaining extract wasstored at −80° C. pending other virological assays.

Postmortem Examinations.

On day 1 p.i., one sham-inoculated dog and one virus-inoculated dog wereeuthanatized by intravenous inoculation of Beuthanasia-D® solution (1mL/5 kg body weight; Schering-Plough Animal Health Corp). Onevirus-inoculated dog was similarly euthanatized each day from days 2 to5 p.i. On day 6 p.i., the remaining sham-inoculated and virus-inoculateddog were euthanatized. Complete postmortem examinations were performedby one of the investigators (WLC). Tissues were fixed in 10% neutralbuffered formalin, embedded in paraffin, and 5-μm sections were eitherstained with hematoxylin and eosin for histopathologic diagnosis orprocessed for immunohistochemistry as described below. Unfixed lungtissues were submitted to the Diagnostic ClinicalMicrobiology/Parasitology/Serology Service at the University of FloridaCollege of Veterinary Medicine for bacterial isolation andidentification. The samples were cultured on nonselective media as wellas media selective for Bordetella species (Regan-Lowe; Remel, Lenexa,Kans.) and Mycoplasma species (Remel). All cultures were held for 21days before reporting no growth. Unfixed tissues were also stored at−80° C. pending virological analyses.

Immunohistochemistry.

Deparaffinized and rehydrated 5-μm trachea and lung tissue sections weremounted on Bond-Rite™ slides (Richard-Allan Scientific, Kalamazoo,Mich.) and subsequently treated with proteinase K (DAKOCytomation Inc.,Carpenteria, Calif.) followed by peroxidase blocking reagent (DAKO®EnVision™ Peroxidase Kit, DAKO Corp., Carpenteria, Calif.). The sectionswere incubated with a 1:500 dilution of monoclonal antibody to influenzaA H3 (Chemicon International, Inc., Ternecula, Calif.) for 2 hours atroom temperature. Controls included incubation of the same sections withmouse IgG (1 mg/mL, Serotec, Inc. Raleigh, N.C.), and incubation of themonoclonal antibody with normal canine lung sections. Followingtreatment with the primary antibody, the sections were incubated withsecondary immunoperoxidase and peroxidase substrate reagents (Dako®EnVision™ Peroxidase Kit, Dako Corp.) according to the manufacturer'sinstructions. The sections were counterstained with hematoxylin, treatedwith Clarifier #2 and Bluing Reagent (Richard-Allan Scientific,Kalamazoo, Mich.), dehydrated, and coverslips applied with Permount(ProSciTech, Queensland, Australia).

RNA Extraction from Swabs and Tissues.

Lung and tracheal tissues from each dog were thawed and homogenized inminimum essential medium (MEM) supplemented with 0.5% bovine serumalbumin (BSA) and antibiotics (gentamycin and ciprofloxacin) using adisposable tissue grinder (Kendall, Lifeline Medical Inc., Danbury,Conn.). Total RNA was extracted from the tissue homogenates as well asorpharyngeal and rectal swab extracts using a commercial kit (RNeasy®Mini Kit, QIAGEN Inc., Valencia, Calif.) according to manufacturer'sinstructions and eluted in a final volume of 60 μL of buffer.

Real-Time RT-PCR.

A single-step quantitative real-time RT-PCR was performed on the totalRNA using the QuantiTect® Probe RT-PCR Kit containing ROX as a passivereference dye (QIAGEN Inc., Valencia, Calif.) and a primer-probe setthat targeted a highly conserved region of the matrix (M) gene of type Ainfluenza virus (Payungporn S. et al., 2006a; Payungporn S. et al.,2006b). For each real-time RT-PCR reaction, 5 μL of extracted total RNAwere added to a reaction mixture containing 12.5 μL of 2X QuantiTech®Probe RT-PCR Master Mix, 0.25 μL of QuantiTech® RT Mix, forward andreverse primers (0.4 μM final concentration for each), probe (0.1 μMfinal concentration) and RNase-free water in a final volume of 25 μL.The TaqMan® GAPDH Control Reagents (Applied Biosystems, Foster City,Calif.) were used according to manufacturer's instructions for detectionof GAPDH as an endogenous internal control for the presence of RNAextracted from the swab and tissue samples and as a normalizationcontrol.

Quantitative one-step real-time RT-PCR was performed on the reactionmixtures in a Mx3000P® QPCR System (Stratagene, La Jolla, Calif.).Cycling conditions included a reverse transcription step at 50° C. for30 minutes, an initial denaturation step at 95° C. for 15 minutes toactivate the HotStarTaq® DNA polymerase, and amplification for 40cycles. Each amplification cycle included denaturation at 94° C. for 15seconds followed by annealing/extension at 60° C. for 1 minute. The FAM(emission wavelength 518 nm) and VIC (emission wavelength 554 nm)fluorescent signals were recorded at the end of each cycle. Thethreshold cycle (Ct) was determined by setting the thresholdfluorescence (dR) at 1000 in each individual experiment. The Mx3000P®version 2.0 software program (Stratagene, La Jolla, Calif.) was used fordata acquisition and analysis. The positive control consisted ofamplification of RNA extracted from A/canine/FL/242/03 (H3N8) virus. Theresults were normalized by dividing the M Ct value by the correspondingGAPDH Ct value for each sample.

Virus Re-Isolation from Tissues.

Frozen lung and trachea tissues from virus-inoculated dogs were thawedand homogenized in 10 volumes of DMEM supplemented with 0.5% BSA andantibiotics. Solid debris was removed by centrifugation and supernatantswere inoculated onto MDCK cells cultured in DMEM supplemented with 1μg/mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, Mo.) andantibiotics as described above. Cells were grown in 25 cm² flasks at 37°C. in a humidified atmosphere containing 5% CO₂. The cultures wereobserved daily for morphologic changes and harvested at 5 days postinoculation. The harvested cultures were clarified by centrifugation andthe supernatants inoculated onto fresh MDCK cells as described for theinitial inoculation; two additional passages were performed for samplesthat did not show evidence of influenza virus by hemagglutination orRT-PCR. Hemagglutination activity in the clarified supernatants wasdetermined using 0.5% turkey red blood cells as previously described(Crawford et al., 2005). RT-PCR was performed as described below.

RT-PCR, Nucleotide Sequencing, and Phylogenetic Analyses.

Viral RNA was extracted from MDCK supernatant using the QIAamp® ViralRNA Mini Kit (QIAGEN Inc., Valencia, Calif.) according to manufacturer'sinstructions. The viral RNA was reverse transcribed to cDNA using theQIAGEN® OneStep RT-PCR Kit (QIAGEN Inc., Valencia, Calif.) according tomanufacturer's instructions. PCR amplification of the coding region ofthe 8 influenza viral genes in the cDNA was performed as previouslydescribed (Crawford et al., 2005), using universal gene-specific primersets (primer sequences available on request). The resulting DNAamplicons were used as templates for automated sequencing in the ABIPRISM® 3100 automated DNA sequencer using cycle sequencing dyeterminator chemistry (Applied Biosystems, Foster City, Calif.).Nucleotide sequences were analyzed using the Lasergene 6 Package®(DNASTAR, Inc., Madison, Wis.). The nucleotide sequences for virusesrecovered from infected dogs were compared to the sequences of the virusin the inoculum to determine if any changes had occurred duringreplication in the respiratory tract.

Example 12 Clinical Disease

All 6 virus-inoculated dogs developed a transient fever (rectaltemperature ≧39° C.) for the first 2 days p.i., but none exhibitedrespiratory signs such as cough or nasal discharge over the 6-dayobservation period. The sham-inoculated dogs remained clinicallyhealthy.

Example 13 Virus Shedding

Influenza A nucleoprotein was detected in the oropharyngeal swabcollected from one of the virus-inoculated dogs at 24 hours p.i. Theoropharyngeal swabs collected from one dog at 72, 84, and 120 hoursp.i., and another dog at 108, 120, and 132 hours p.i., were positive forvirus by quantitative real-time RT-PCR (Table 11). The absolute numberof influenza M gene copies per μL of swab extract increased with timefrom 3 to 6 days p.i. No virus was detected in the rectal swabs.

Example 14 Postmortem Examinations

In contrast to the previous experimental infection using specificpathogen-free Beagles (Crawford et al., 2005), the virus-inoculatedmongrel dogs had pneumonia as evidenced by gross and histologicalanalyses of the lungs from days 1 to 6 p.i. In addition to pneumonia,the dogs had rhinitis, tracheitis, bronchitis, and bronchiolitis similarto that described in naturally infected dogs (Crawford et al., 2005).There was epithelial necrosis and erosion of the lining of the airwaysand bronchial glands with neutrophil and macrophage infiltration of thesubmucosal tissues (FIG. 5, upper panels). Immunohistochemistry detectedviral H3 antigen in the epithelial cells of bronchi, bronchioles, andbronchial glands (FIG. 5, lower panels). No bacterial superinfection waspresent. The respiratory tissues from the 2 sham-inoculated dogs werenormal.

Example 15 Virus Replication in Trachea and Lungs

The trachea and lungs were positive for virus by quantitative real-timeRT-PCR in all dogs from 1 to 6 days p.i. (Table 12). The absolute numberof influenza M gene copies per μL of trachea homogenate increased from 1to 5 days p.i., then decreased on day 6. The absolute number of M genecopies per μL of lung homogenate decreased from 1 to 6 days p.i. Ingeneral, the trachea contained ≧one log₁₀ more virus than the lung oneach of the 6 days p.i.

TABLE 11 Detection of virus shedding in the oropharynx of mongrel dogsinoculated with canine influenza virus by quantitative real-time RT-PCR.Dog Time p.i. M/GAPDH Matrix gene ID (hours)^(a) ratio^(b)(copies/uL)^(c) 860 72 1.20 1.57E+02 84 1.30 8.25E+02 120 1.23 1.47E+03894 108 1.17 1.17E+02 120 1.41 1.37E+02 132 1.27 3.74E+02 ^(a)Time thatoropharyngeal swabs were collected from the dogs following inoculationwith A/canine/FL/43/04 (H3N8) virus. ^(b)Normalization ratios werecalculated by dividing the M (Ct) by the GAPDH (Ct) for each swabextract. ^(c)The absolute number of matrix gene copies per uL of swabextract.

TABLE 12 Detection of virus replication in the trachea and lung ofmongrel dogs inoculated with canine influenza virus by quantitativereal-time RT-PCR. M/GAPDH Matrix gene Dog Time p.i. ratio^(b)(copies/uL)^(c) ID (hours)^(a) Lung Trachea Lung Trachea 797 24 1.201.43 8.22E+05 3.11E+04 801 48 1.33 0.99 1.15E+05 6.52E+06 789 72 1.441.12 2.39E+04 1.56E+05 819 96 1.40 1.27 3.19E+04 1.43E+05 860 120 1.591.04 3.48E+03 1.17E+06 894 144 1.70 1.15 4.78E+02 1.50E+03 ^(a)Time thattissues were collected from the dogs following inoculation withA/canine/FL/43/04 (H3N8) virus. ^(b)Normalization ratios were calculatedby dividing the M (Ct) by the GAPDH (Ct) for each tissue homogenate.^(c)The absolute number of matrix gene copies per uL of tissuehomogenate.

Materials and Examples Methods for Example 16 Virus Strains

Canine influenza virus strains as well as those of avian, equine andhuman origin (listed in Table 15) were propagated in embryonated eggs orMDCK cells and their infectivity was titrated by endpoint dilution inchicken embryos, or plaque assay. Rapid virus quantification wasperformed by hemagglutination assay using turkey red blood cellerythrocytes.

Diagnostic Specimens

A Total of 60 canine's lung tissues collected from suspect cases ofviral respiratory disease during the year of 2005 were tested for thepresence of canine influenza virus.

RNA Extraction from Canine Tissue Samples

Blocks of lung tissue weighing between 20 and 30 mg were homogenized ina disposable tissue grinder (Kendal). Total RNA was extracted using acommercial kit (RNeasy Mini Kit, Qiagen, Valencia, Calif.) and eluted ina final volume of 60 μL, following the manufacturer's recommendations.

Primers and Probes Design

Multiple sequence alignments of the H3 and M genes from various subtypesand from diverse species were performed using the CLUSTAL X program(Version 1.8). Matrix (M) primers and probe were selected from theconserved regions of over the known sequences corresponding to differentsubtypes of influenza A virus, whereas the H3 hemagglutiningene-specific primers and probe set were selected to specifically matchequine and canine influenza A virus genes and mismatch the homologousavian and human genes (Table 13). Primer design software (OLIGOS Version9.1) and the web based analysis tools provided by EXIQON(http://lnatools.com) was used for Tm calculation and prediction ofsecondary structure as well as self hybridization. A conserved region ofan 18S rRNA gene was used as endogenous internal control for thepresence of RNA extracted from canine tissue sample. The Pre-DevelopedTaqMan® Assay Reagents for Eukaryotic 18S rRNA (VIC/TAMRA) (AppliedBiosystems) was used for the real-time detection of 18S rRNA in tissuesamples.

Real-Time RT-PCR Condition

A single-step real-time RT-PCR was performed by using the QuantitectProbe RT-PCR Kit containing ROX as a passive reference dye (Qiagen,Valencia, Calif.). In each real-time RT-PCR reaction, 5 μL of RNA samplewere used as a template to combine with a reaction mixture containing 10μL of 2X QuantiTech Probe RT-PCR Master Mix, 0.2 μL of QuantiTech RTMix, primers (0.4 μM final conc. for H3 gene or 0.6 μM final conc. for Mgene), probe (0.1 μM final conc. for H3 gene or 0.2 μM final conc. for Mgene) and RNase-free water in a final volume of 20 μL. One-stepreal-time RT-PCR was performed in the Mx3005P Real-Time QPCR System(Stratagene). Cycling conditions included a reverse transcription stepat 50° C. for 30 minutes. After an initial denaturation step at 95° C.for 15 minutes in order to activate the HotStarTaq DNA polymerase,amplification was performed during 40 cycles including denaturation (94°C. for 15 seconds) and annealing/extension (60° C. for 30 seconds). TheFAM (emission wavelength 516 nm for H3 and M detection) and VIC(emission wavelength 555 nm for 18S rRNA detection) fluorescent signalswere obtained once per cycle at the end of the extension step. Dataacquisition and analysis of the real-time PCR assay were performed usingthe Mx3005P software version 2.02 (Stratagene).

Specificity of H3 Primers/Probe Set for Canine Influenza (H3N8) andUniversality of M Primers/Probe Set for Type a Influenza Virus

In order to test the specificity of each primers/probe set, RNAextracted from several known subtypes of influenza A viruses were usedas a template in the real-time RT-PCR assay (Table 15).

RNA Standard for Determination of the Real-Timer RT-PCR Performance

The genes of canine influenza A virus (A/canine/Florida/242/2003(H3N8))were used to generate the PCR amplicons for H3 (nt 1-487) and M (nt1-276) by using primers linked with T7 promoter (Table 13). Then thepurified PCR amplicons of H3 and M genes were used as templates for invitro transcription by using Riboprobe in vitro Transcription System-T7(Promega) following the manufacturer's recommendations. Theconcentration of the transcribed RNAs was calculated by measuringabsorbance at 260 nm. The RNAs were then serially diluted 10-fold,ranging from 10⁸ to 10 copies/μL to perform sensitivity tests. Moreover,a standard curve was generated by plotting the log of initial RNAtemplate concentrations (copies/μL) against the threshold cycle (Ct)obtained from each dilution in order to determine the overallperformance of real-time RT-PCR.

Comparative Sensitivity Tests Between Real-Time RT-PCR and DirectigenFlu a Test Kit

Stock viruses of two viral strains including A/Wyoming/3/2003 (H3N2) at10^(6.67) EID₅₀/mL (HA=64) and A/canine/Florida/242/2003(H3N8) at10^(7.17) EID₅₀/mL (HA=16) were used for the detection threshold assay.Logarithmic dilution of specimens in phosphate-buffered saline (PBS)(125 μL) were used in a rapid influenza A antigen detection kit,Directigen Flu A, (Becton, Dickinson and Company) following themanufacturer's instructions. Each Directigen Flu A test device has anH1N1 influenza antigen spot in the center of the membrane which developsas a purple dot and indicates the integrity of the test, which is basedon a monoclonal antibody to the nucleoprotein (NP). The development of apurple triangle surrounding the dot is indicative of the presence ofinfluenza NP in the tested specimen. The intensity of the purple signalfrom the triangle was scored as +(outline of triangle), ++(lightlycolored triangle), +++(dark-purple triangle) and ++++(very dark-purpletriangle). Viral RNA was extracted 125 μL aliquots of each virusdilution by using QIAamp Viral RNA Mini Kit (Qiagen, Valencia, Calif.)and eluting in a final volume of 50 μL. A volume of 5 uL of theextracted viral RNAs were tested by real-time RT-PCR for comparativesensitivity test with Directigen Flu A kit.

Example 16

The real-time RT-PCR assay for canine influenza relies on informationfrom three molecular probes which target 18S rRNA from host cell waswell as M and H3 from the influenza A virus genome (Table 14).Amplification of the host gene is a reporter of specimen quality andintegrity. Clinical, necropsy or laboratory samples containing canineinfluenza (H3N8) virus are expected to yield amplification signal withthe three probes. Specimens yielding amplification signal with M and 18SrRNA probes but negative for H3 would be indicative of an influenzavirus subtype H3 from human, swine or avian origin or from non-H3subtypes. These rare cases could be resolved by RT-PCR using HAuniversal primers to generate amplicon cDNA that can be analyzed bysequencing. Properly collected and handled specimens lacking influenza Avirus yield 18S rRNA amplification signal only. Situations in which onlythe 18S rRNA probe and the H3 probes yield amplification signal areindicative of faulty technique, unless proven otherwise; either a falsenegative with M probes or false positive for H3 need to be demonstrated.Finally, specimens failing to yield amplification signals with the threeprobes are indicative of defective sample collection, degradation,faulty RNA extraction or the presence of inhibitors the polymerases usedin PCR.

In order to test the specificity of the H3 primers/probe set for canineinfluenza A virus (H3N8) and the universality of M primers/probe set fortype A influenza, several subtypes of influenza A viruses were tested byreal-time RT-PCR. The results show that H3 primers/probe set yielded apositive amplification signal only with canine influenza (H3N8). Nosignificant false positive or non-specific amplification signals wereobserved in other subtypes or human H3 strains. The M primers/probe setyielded positive amplification signal with all of the strains tested(Table 15). These results indicated that H3 primers/probe specificallydetects canine influenza A virus (H3N8) whereas M primers/probe detectmultiple subtypes of type A influenza viruses.

The performance of real-time RT-PCR assays was evaluated by endpointdilution of M and H3 in vitro transcribed RNAs. As expected, thethreshold cycle (Ct) increased in direct correlation with the dilutionof the RNA standards. The fluorescent signals can be detected at RNAstandard dilutions of M and H3 as low as 10³ and 10² copies/μL,respectively (FIGS. 6A and 6B). The standard curves of M and H3 geneswere constructed by plotting the log of starting RNA concentrationsagainst the threshold cycle (Ct) obtained from each dilution (FIGS. 6Cand 6D). The slope of the standard curve is used to determine the PCRreaction efficiency, which is theoretically exponential; 100%amplification efficiency would imply doubling of amplicon concentrationeach cycle. The standard curves with a slope between approximately −3.1and −3.6 are typically acceptable for most applications requiringaccurate quantification (90-110% reaction efficiency). An Rsq value isthe fit of all data to the standard curve plot. If all the data lieperfectly on the line, the Rsq will be 1.00. As the data fall furtherfrom the line, the Rsq decreases. An Rsq value ≧0.985 is acceptable formost assays. The M standard curve revealed a slope of −3.576(efficiency=90.4%) and Rsq=1.00 whereas H3 standard curve yielded aslope of −3.423 (efficiency=95.9%) and Rsq=0.999. These values indicatesatisfactory amplification efficiency and overall performance of thereal-time RT-PCR assays. We attribute the lower efficiency andsensitivity of M primers/probe set as compared to H3 primers/probe setto the N-fold degeneracy of M primer sequences required to ensure broadcoverage of M gene sequences variability across viruses of multiplesubtypes, hosts and lineages.

The sensitivity of real-time RT-PCR assay was also compared with thecommercial rapid antigen detection assay (Directigen Flu A). Logarithmicdilutions of A/Wyoming/3/2003 (H3N2) and A/canine/Florida/242/2003(H3N8)were analyzed with Directigen Flu A and by real-time RT-PCR. The resultsof Directigen Flu A showed that the sensitivities against both viralstrains are approximately 100-fold dilution from the stock viruses usedin these experiments (FIG. 7). The signals (purple color) generated bythe canine virus (A/canine/Florida/242/2003: 10^(6.x) PFU/ml) sampleswere much weaker than those found in human virus (A/Wyoming/3/2003:10^(7.x) PFU/ml), in agreement with the lower virus concentration inthese samples. Alternatively, lower signal for canine influenza could beattributed to the molecular specificity of monoclonal antibodies againstthe NP; i.e. poor conservation of the amino acids within the NP epitopeof canine influenza A viruses.

Real-time RT-PCR of the M gene yielded Ct values above threshold withvirus 10 and 30 PFU equivalents per reaction ofA/canine/Florida/242/2003 and A/Wyoming/3/2003, respectively (Table 16).The differences between the sensitivity value of 2 viral strains becausethe differences in the original viral titers. The H3 gene detectioncomparison between canine and human influenza viruses was not performedbecause the H3 primers/probe in our realtime RT-PCR assay amplifiesexclusively canine influenza A virus. RT-PCR was 10⁵ times moresensitive than the rapid antigen detection kit.

To evaluate the performance of the RT-PCR test in necropsy specimensfrom dogs with acute respiratory disease, sixty canine lung tissuesamples submitted during the year of 2005 were tested for the presenceof canine influenza A virus by real-time RT-PCR. A total of 12 out of 60samples (20%) were positive with both M and H3 genes whereas theremaining 48 samples yielded negative result for both M and H3 gene.Virus isolation attempts were conducted by egg and MDCK cellinoculation, to evaluate the specificity of the realtime assay; 2 out 12samples that were positive for canine influenza by RT-PCR yielded canineinfluenza virus (data not shown, manuscript in preparation). Althoughall of the tissues were collected from dogs with a history of severerespiratory disease, most of the samples yielded no canine influenzavirus by either realtime PCR or conventional isolation, suggesting ahigh incidence of other respiratory pathogens such as Bordetellabronchiseptica, canine distemper or parainfluenza virus. The single stepreal-time RT-PCR assay herein provides a rapid, sensitive andcost-effective approach for canine influenza A virus (H3N8) detection.Rapid laboratory diagnosis of canine influenza A virus (H3N8) infectionsin the early stage of the disease can yield information relevant toclinical patient and facility management.

TABLE 13Primers and probes used for real-time RT-PCR detection and in vitro transcriptionOligo name Type Target Sequence* Application Ca-H3-F387 ForwardH3 (nt 387-406) 5′-tatgcatcgctccgatccat-3′ Real-time primer(SEQ ID NO: 79) PCR Ca-H3-R487 Reverse H3 (nt 487-467)5′-gctccacttcttccgttttga-3′ primer (SEQ ID NO: 80) Ca-H3-P430 TaqManH3 (nt 430-459) FAM-aattcacagcagagggattcacatggacag-BHQ1 probe(SEQ ID NO: 81) FluA-M-F151 Forward M (nt 151-174) 5′-catggartggctaaagacaagacc-3′ Real-time primer (SEQ ID NO: 82) PCRFluA-M-R276 Reverse M (nt 276-253) 5′-agggcattttggacaaakcgtcta-3′ primer(SEQ ID NO: 83) FluA-M-P218 LNA M (nt 218-235)5′-acgcTcaccgTgcccAgt-BHQ1 TaqMan (SEQ ID NO: 84) probe H3-F1 ForwardH3 (nt 1-14) 5′-tattcgtctcagggagcaaaagcagggg-3′ In vitro primer(SEQ ID NO: 85) transcription T7/H3-R490 Reverse T7/H3 (nt 487-467)5′-tgtaatacgactcactatagggctccacttcttccgttttga-3′ primer (SEQ ID NO: 86)M-F1 Forward M (nt 1-15) 5′-gatcgctcttcagggagcaaaagcaggtag-3′ In vitroprimer (SEQ ID NO: 87) transcription T7/M-R276 Reverse M (nt 276-253)5′-tgtaatacgactcactatagggcattttggacaaagcgtc-3′ primer (SEQ ID NO: 88)*Note: Uppercases = LNA (Locked Nucleic Acid) residues, r = a or g, k =g or t, underline = T7 promoter sequence

TABLE 14 Interpretation of the real-time RT-PCR assay ResultsInterpretation M H3 18S rRNA Positive for canine influenza A virus(H3N8) + + + Positive for influenza A virus (unknown subtype) + − +Negative for influenza A virus − − + Error in RNA extraction or presenceof PCR − − − inhibitor

TABLE 15 Specificity test of canine H3 primers/probe set anduniversality test of M primers/probe set with several subtypes ofinfluenza A viruses Real-time RT-PCR detection H3 M Sub- gene gene typesStrain Name Host (Ct) (Ct) H1 A/Ohio/1983 Human No Ct 15.40 A/WSN/1933Human No Ct 20.09 H3 A/Wyoming/3/2003 Human No Ct 28.85A/Victoria/3/1975 Human No Ct 16.62 A/canine/FL/242/2003 Canine 28.4329.25 H4 Turkey/MN/1066/1980 Avian No Ct 17.49 Clinical sample* Avian NoCt 20.87 H5 AChicken/Thailand/CUK2/2004 Avian No Ct 20.13A/Pheasant/NJ/1335/1998 Avian No Ct 16.64 H6 Clinical sample* Avian NoCt 19.52 H10 Clinical sample* Avian No Ct 25.64 Clinical sample* AvianNo Ct 19.59 H11 Clinical sample* Avian No Ct 15.72 Clinical sample*Avian No Ct 24.55 *Note that subtypes of clinical samples were confirmedby nucleotide sequencing.

TABLE 16 Comparative sensitivity tests for influenza A virus detectionbetween real-time RT-PCR and Directigen Flu A Directigen Flu A Real-timeRT-PCR of M (Ct) Virus A/canine/ A/Wyoming/ A/canine/ A/Wyoming/dilutions 242/03 3/03 242/03 3/2003 10⁻¹ + + + + + + 22.42 19.4810⁻² + + + + 25.85 22.66 10⁻³ − − 29.27 25.76 10⁻⁴ Not done Not done32.66 28.66 10⁻⁵ Not done Not done 35.48 33.14 10⁻⁶ Not done Not done37.51 35.06 10⁻⁷ Not done Not done 39.09 36.44 10⁻⁸ Not done Not done NoCt 38.93

TABLE 17 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

TABLE 18 Letter Symbol Amino Acid A Alanine B Asparagine or asparticacid C Cysteine D Aspartic Acid E Glutamic Acid F Phenylalanine GGlycine H Histidine I Isoleucine K Lysine L Leucine M Methionine NAsparagine P Proline Q Glutamine R Arginine S Serine T Threonine VValine W Tryptophan Y Tyrosine Z Glutamine or glutamic acid

TABLE 19 Amino acid differences between PB2 proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 5 K K E 12 S L L 37 G G E 175 R R I 374 L I I 375 R R K 447Q Q H

TABLE 20 Amino acid differences between PB1 proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 114 V I I 154 D G G 221 A T T 317 M I I 459 I I V 682 I I V

TABLE 21 Amino acid differences between PA proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 27 D N N 62 I V V 213 R K K 337 A T T 343 A E E 345 L I I353 K R R 400 T T A 450 V I I 460 M M I 673 R R K 675 N D D * Based onavailable genes of viruses isolated between 1963 and 1998.

TABLE 22 Amino acid differences between NP proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 16 G D D 157 A T T 214 R R K 285 V V I 286 A T T 359 A T T375 D D N 384 R K K 452 R K K

TABLE 23 Amino acid differences between NA proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 9 A/T T A 12 S F F 20 L I I 40 G R R 42 G D D 46 N K K 52 EE K 61 R K K 69 N S S 72 E K K 201 V I I 261 I V V 301 I I V 396 N D D397 L P P

TABLE 24 Amino acid differences between M1 proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 M1 161 S S A M1 208 K/Q R R * Based on available genes ofviruses isolated between 1963 and 1998.

TABLE 25 Amino acid differences between NS1 proteins of H3N8 equine andcanine influenza viruses Position Equine Consensus * Canine/FL/03Canine/FL/04 44 K R R 59 R H H 71 E K K 86 A T T 88 R R L 140 R G G 216P S S * Based on available genes of viruses isolated between 1963 and1998.

Example 17 Canine Influenza Challenge Model Development

The canine influenza (canine flu) virus, which was isolated from fluoutbreaks in Florida, was observed to be a H3N8 type influenza virus,and closely related to equine flu virus strain, A/equine/Ohio/03(Crawford et al., SCIENCE Vol. 309, September 2005, incorporated byreference in its entirety into this patent). The potential of using theequine flu virus strain A/equine/Ohio/03 to induce influenza-likedisease in dogs was investigated in this study.

Procedure:

Ten 13-week-old beagles of mixed sex were obtained from a commercialsupplier, and housed in individual cages in a BSL-2 facility. The dogswere randomly assigned to two groups of 5 dogs each. As shown in Table26, one group was subjected to a intratracheal challenge, and the othergroup was subjected to an oronasal challenge. The dogs were challengedat 14 weeks-of-age.

TABLE 26 Experimental Design Group Number of Dogs Challenge Route 1 5Intratracheal 2 5 Oronasal

A cell culture grown equine flu virus A/equine/Ohio/03 was used as thechallenge virus. For intratracheal challenge, the challenge virus wasadministered via a delivery tube, which consisted of a cuffed trachealtube (Size 4.0/4.5, Sheridan, USA) and feeding tube (size 5 Fr, 1.7 mm,/16 inches in length, Kendall, USA) in 0.5 to 1.0 ml volume. Fororonasal challenge, the challenge virus (10⁷ to 10⁸ TCID50 per dog) wasadministered as a mist using a nebulizer (DeVilbiss ULTRA-NEB 99ultrasonic nebulizer, Sunrise Medical, USA) in a 2 to 3 ml volume.

The dogs were observed for flu related clinical signs for 14 dayspost-challenge. Serum samples were collected from each dog on day zero(before challenge), and days 7 and 14 post-challenge for determining theHI titer using a H3N8 equine influenza virus with a standard protocol(SAM 124, CVB, USDA, Ames, Iowa). All dogs were humanely euthanized andlung tissues were collected in 10% buffered formalin forhistopathological evaluation.

Results:

The results of this experiment are summarized in Table 27. Influenzarelated clinical signs were observed in a few dogs after challenge.These signs included fever (>103° F.; >39.4° C.) and cough. Two of 5dogs (i.e., 40%) had fevers (>103° F.; >39.4° C.) in Group 1, comparedto 1 of 5 (i.e., 20%) dogs in Group 2. One dog from the oronasalchallenge group had sneezing, and another had cough following thechallenge. An HI titer range from 10 to 80, with a geometric mean titer(GMT) of 20, was observed for Group 1. A titer range of 40 to 160, witha GMT of 86, was observed for Group 2. One dog from each group hadhistopathological lesions compatible with or pathognomic for influenza.

TABLE 27 Canine flu challenge - clinical signs, virus isolation,histopathology results and serology results Serology (HI titer) Virusisolation Microscopic 7-days 14-days Dog* Challenge Clinical Nasal/oralTracheal Lung lesion Pre- post post ID method* signs swab scrapingtissues (histopathology) challenge challenge challenge AAH Intratrachealnone negative negative negative negative 10 10 20 ADB Intratracheal nonenegative negative negative negative 10 80 20 ADC Intratracheal Fever*negative negative negative negative 10 20 20 AEB Intratracheal Fevernegative negative negative positive 10 40 20 AEE Intratracheal nonenegative negative negative inconclusive 10 20 10 AAE Oronasal nonenegative negative negative negative 10 80 80 AAG Oronasal none negativenegative negative negative 10 40 80 ABY Oronasal Occasional negativenegative negative positive 10 80 160 sneeze, occasional cough ADYOronasal Fever, negative negative negative negative 10 80 80 occasionalsneeze ADZ Oronasal none negative negative negative negative 10 80 160*The animals were challenged with an Equine flu isolate Ohio 03.**Rectal temperature ≧103° F.; ≧39.4° C.

Example 18 Efficacy of an Equine Influenza Virus Vaccine for Dogs

The canine influenza (canine flu) virus isolated from flu outbreaks inFlorida was observed to be a H3N8 type influenza virus, and was closelyrelated to equine flu virus, A/equine/Ohio/03 based on the sequencesimilarity. The following study was conducted to determine the efficacyof an experimental inactivated equine influenza virus vaccine.

Procedure:

Nine 7-week-old beagles of mixed sex were obtained from a commercialsupplier, and housed in individual cages in a BSL-2 facility. These dogswere randomly assigned to two groups, as summarized in Table 28:

TABLE 28 Experimental Design Group Number of Dogs Treatment 1 5 Vaccine2 4 ControlThe first group consisted of 5 dogs, which were vaccinated with aninactivated, CARBIGEN™ (MVP Laboratories, Inc., Omaha, Nebr.)adjuvanted, equine flu virus A/equine/Ohio/03 vaccine at 8 and 12weeks-of-age via subcutaneous (SQ) route. The A/equine/Ohio/03 wasinactivated by binary ethylenimine (“BEI”) using a standard method. Eachdose of the vaccine contained 5% by mass CARBIGEN™, 4096 HA units of theinactivated virus, sufficient PBS to bring the total volume of the doseto 1 ml, and sufficient NaOH to adjust the pH to between 7.2 and 7.4.Serum samples were collected from all dogs on the day of first andsecond vaccination and day 7 and 14, post-first and -second vaccination,and at pre-challenge for determining the HI titer using a H3N8 equineinfluenza virus a standard protocol (SAM 124, CVB, USDA, Ames, Iowa). At3 weeks post-second vaccination, the 5 vaccinated dogs and the secondgroup (i.e., the control group) consisting of 4 age-matched dogs werechallenged oronasally with a cell-culture-grown equine influenza virusA/equine/Ohio/03 (10^(7.0) to 10^(8.0) TCID50 per dog) in a 1-2 mlvolume per dose. The challenge virus was administered to the dogs as amist using a nebulizer (DeVilbiss ULTRA-NEB 99 ultrasonic nebulizer,Sunrise Medical, USA). The dogs were observed for flu-related clinicalsigns for 14 days post-challenge. Five dogs (3 vaccinates and 2controls) 7 days post-challenge and 4 dogs (2 controls and 2 vaccinates)14 days post-challenge were humanely euthanized for collection of lungtissues in 10% buffered formalin for histopathological evaluation.

Results:

The results of this experiment are summarized in Tables 29 and 30. Allvaccinated dogs seroconverted following the vaccination. An HI titerrange from 40 to 640, with the GMT of 129, was observed during thepost-vaccination period with equine influenza virus A/equine/Ohio/03,and a HI titer of 160 to 320, with a geometric mean titer of 211, wasobserved with canine flu isolate, A/canine/Florida/242/03. Two of 6vaccinates had a fever of >103° F. (>39.4° C.) for one day and no otherclinical signs were observed in any of the dogs following challenge.

Conclusion:

All the vaccinated dogs responded to the inactivated, CARBIGEN™adjuvanted equine influenza vaccine. The HI titer results with a canineinfluenza virus isolate suggest that the inactivated equine influenzavaccine did induce a detectable level of cross reactive antibody tocanine influenza virus. Even though the challenge virus used in this didnot induce any noticeable clinical disease in beagle dogs, based on theHI titer with a canine influenza virus isolate, it was concluded thatinactivated equine vaccine could be used in dogs to induce crossreactive antibodies, which could potentially protect dogs against“canine flu” disease caused by H3N8 type canine influenza viruses.

TABLE 29 Serology - Pre- and post-vaccination and post-challenge HItiters HI titers Post-1^(st) Post-2^(nd) Pre- vaccination vaccinationPost-challenge* Dog* Group vaccination 7-d 14-d 7-d 14-d 21-d 7-d 14-dAKT Vaccinate** <10 40 80 640 640 640 320 320  ALH Vaccinate** <10 40 80320 160 160 80 *** ALU Vaccinate** <10 40 80 320 160 160 80 80 ANJVaccinate** <10 40 80 320 160 80 320 *** ANU Vaccinate** <10 40 80 320160 80 160 *** AJW Control <10 <10 <10 <10 <10 <10 10 *** AKR Control<10 <10 <10 <10 <10 <10 10 *** ALZ Control <10 <10 <10 <10 <10 <10 20 20ARC Control <10 <10 <10 <10 <10 <10 10 10 *The animals were challengedwith an equine flu isolate Ohio 03 **CARBIGEN ™ adjuvanted inactivatedequine flu virus Ohio 03 vaccine was used for vaccination *** Euthanized7-days post-challenge

TABLE 30 Canine flu challenge* - clinical signs, virus isolation,histopathology results Clin- Virus isolation Microscopic Dog Treatmentical Nasal Tracheal Lung lesion (his- ID group signs swab scrapingtissues topathology) AKT Vaccinate** none negative negative negativenegative ALH Vaccinate** none negative negative negative negative ALUVaccinate** none negative negative negative negative ANJ Vaccinate**none negative negative negative negative ANU Vaccinate** none negativenegative negative negative AJW Control none negative negative negativenegative AKR Control none negative negative negative negative ALZControl none negative negative negative negative ARC Control nonenegative negative negative negative *The animals were challenged with anEquine flu isolate Ohio 03 **CARBIGEN ™ adjuvanted inactivated equineflu virus Ohio 03 vaccine was used for vaccination

Example 19 Efficacy of an Equine Influenza Virus Vaccine for Dogs

The canine influenza virus isolated from flu outbreaks in Florida wascharacterized is closely related to a number of H3N8 type equineinfluenza virus isolates. By DNA and amino acid sequence similarityanalysis it was demonstrated that the canine influenza virus is verysimilar to an equine influenza virus, A/equine/Ohio/03. The followingstudy was conducted in dogs to determine the efficacy of commerciallyavailable equine influenza vaccines in dogs.

Procedure:

Approximately 16 month old, 20 mongrels and 20 beagles of mixed sex wereused in the study. The dogs were randomly assigned to 6 groups (Table31) of 6-7 dogs each. Dogs in groups 1 and 4 were vaccinated with acommercially available inactivated, adjuvanted equine influenza vaccine(EQUICINE™, Intervet Inc., Millsboro, Del.) at 16 and 17 months of agevia subcutaneous (SQ) route. The dogs in groups 2 and 5 were vaccinatedwith a modified live equine/Kentucky/91 influenza vaccine in a 1 mlvolume via intranasal route (single nostril). Blood samples werecollected on the day of vaccination, day 7 and 14 post first vaccination(groups 1, 2, 4, and 5) and post second vaccination (groups 1 and 4) fordetermining the HI titer using an H3N8 equine influenza virus and acanine influenza virus using per a standard protocol (SAM 124, CVB,USDA, Ames, Iowa).

Vaccinates (at 72 days post final vaccination) and the controls werechallenged oronasally with a cell-culture grown equine influenza virusstrain A/equine/Ohio/03 (10^(7.0) to 10^(8.0) TCID50 per dog) in a 1-2ml volume. The challenge virus was administered to the dogs as mistusing a nebulizer (DeVilbiss ULTRA-NEB 99 ultrasonic nebulizer, SunriseMedical, USA). The dogs were observed for influenza-related clinicalsigns for 12 days post-challenge. The nasal and oropharyngeal swabs werecollected in Earl's MEM medium with antibiotics (neomycin and polymyxinB) daily from day −1 to day 12 post challenge for virus isolation. Thepresence of virus in the swabs indicates that the animal is excretingthe virus in nasal/oral secretions. All dogs were humanely euthanized onday 12 post-challenge and lung tissues were collected in 10% bufferedformalin for histopathological evaluation.

TABLE 31 Experimental design Number Number Route of Group of dogs BreedTreatment of doses vaccination 1 7 Beagle EQUICINE 2 Subcutaneous II ™**2 7 Beagle A/KY/91*** 1 Intranasal 3 6 Beagle Control N/A* N/A* 4 7Mongrel EQUICINE 2 Subcutaneous II ™ 5 7 Mongrel A/KY/91 1 Intranasal 66 Mongrel Control N/A* N/A* *Not applicable **EQUICINE II ™ is marketedby Intervet Inc. as a liquid vaccine. EQUICINE II ™ contains inactivatedA/Pennsylvania/63 influenza (or “A/Pa/63”) virus andA/equine/Kentucky/93 influenza (or “A/KY/93”) virus with carbopol (i.e.,HAVLOGEN ® (Intervet Inc.)). More specifically, a dose of EQUICINE II ™contains: inactivated A/Pa/63 at 10^(6.0) EID₅₀, inactivated A/KY/93 at10^(6.7) EID₅₀, 0.25% by volume carbopol, and sufficient PBS to create atotal volume of 1 ml. ***A/KY/91 is a freeze-dried vaccine that wasreconstituted with water. Such reconstitution was conducted usingvaccine-grade water sufficient to bring the vaccine dosage to a totalvolume of 1 ml. The vaccine contained equine/Kentucky/91 influenza (or“A/KY/91”) virus, and is discussed in, for example, U.S. Pat. Nos.6,436,408; 6,398,774; and 6,177,082, which are incorporated by referencein their entirety into this patent. When reconstituted, a dose of thevaccine contained A/KY/91 at 10^(7.2) TCID₅₀ per ml, 0.015 grams N-ZAMINE AS ™ per ml, 0.0025 grams gelatin per ml, and 0.04 grams D lactoseper ml. N-Z AMINE AS ™ is a refined source of amino acids and peptidesproduced by enzymatic hydrolysis of casein. N-Z AMINE AS ™ is marketedby Kerry Bio-Science (Norwich, NY, USA).

Results:

All vaccinated dogs seroconverted following the vaccination and the HItiters ranged from 10 to 80 for EQUICINE II™ vaccine group dogs comparedto 10 to 40 for the A/KY/91 vaccine group dogs using an equine influenzavirus (H3N8 type).

The samples collected at 2 weeks post vaccination (post secondvaccination for EQUICINE II™ vaccine) were analyzed for HI titerdetermination with a canine influenza as well as with an equineinfluenza virus (H3N8 type). The HI results are shown in Table 32. Theclinical signs include fever (>103° F.; >39.4° C.), occasional cough,and mild nasal discharge observed following the challenge.

TABLE 32 Serology - HI titers at 2 weeks post vaccination HI titer withEquine Canine Number influenza influenza of Treat- virus virus Groupdogs Breed ment Range GMT Range GMT 1 7 Beagle Equicine 10-80 36 10-8033 II ™ 2 7 Beagle A/KY/91 10-20 12  20-160 54 3 6 Beagle Control N/A*N/A* N/A* N/A* 4 7 Mon- Equicine 40-80 54 40-80 50 grel II ™ 5 7 Mon-A/KY/91 10-40 24 40-80 49 grel 6 6 Mon- Control N/A* N/A* N/A* N/A* grel*Not applicable

Among beagles, 2 of 6 dogs in the EQUICINE II™ vaccine group (Group 1),1 of 7 dogs in the A/KY/91 vaccine group (Group 2) and 2 of 6 dogs inthe control group (Group 3) had fever. One of 6 dogs in Group 3(control) was positive for virus in the cell culture supernatant ofnasal swab material by hemagglutination assay with 0.25% chicken redblood cells (CRBC). One of 6 dogs in the control group (Group 3) and 1of 7 dogs in the A/KY/91 vaccine group (Group 2) had mild nasaldischarge during the post challenge observation period. There was nostatistical significant difference (P>0.05) between control and vaccinegroups for beagle dogs.

Among mongrels, 5 of 7 dogs in the EQUICINE II™ vaccine group (Group 4),1 of 7 dogs in the A/KY/91 vaccine group (Group 5) and 5 of 6 dogs inthe control group (Group 6) had fever. One dog from each of Group 4 and6 had a mild nasal discharge, and one dog from Group 5 had an occasionalcough. Two of 7 dogs in the EQUICINE II™ vaccine group (Group 4) and 3of 6 dogs in the control group (Group 6) were positive for influenzavirus in the nasal swab by HA assay. None of the dogs from the A/KY/91group (Group 5) were positive for influenza virus in the nasal swabmaterials.

Conclusion:

By serology, it was demonstrated that vaccination of dogs withcommercially available equine influenza vaccines stimulated a moderatelevel influenza antibody response. There may be some breed difference indevelopment of influenza-related clinical signs in dogs following achallenge with H3N8 type influenza virus. The live attenuated equineinfluenza vaccine (A/KY/91) provided a significant (P<0.05) protectionfrom clinical disease development in rectal temperature in mongrels.Also, the live attenuated viral vaccine prevented the shedding ofinfluenza virus in the nasal secretions.

Example 20 Canine Influenza Challenge Model Development

In view of reports that inducing disease in canines for purposes ofstudy had not proven successful, the potential for using a canineinfluenza virus, H3N8, to develop a canine influenza challenge model indogs was investigated in the following study.

Procedure:

Ten mongrels of mixed sex were obtained from a commercial supplier, andhoused in cages in a BSL-2 facility. The dogs were randomly assigned totwo groups of 5 dogs each. As shown in Table 33, one group was subjectedto an intratracheal/intranasal challenge, and the other group wassubjected.

TABLE 33 Experimental design Group Number of dogs Challenge route 1 5Intratracheal/intranasal 2 5 OronasalThe dogs were challenged at approximately 12 weeks-of-age.Embryonated-chicken-egg grown canine influenza virus(A/canine/Florida/242/03) virus was used as challenge virus. Each dogreceived a total of approximately 10^(7.2) TCID50 of virus in either 2ml (for oronasal route) or 4 ml (intratracheal/intranasal route) volume.

For intratracheal/intranasal challenge, 3 ml of the challenge virus wasadministered into the trachea first, followed by 5 ml of PBS using adelivery tube, which consisted of a cuffed tracheal tube (Size 4.5/5.0,Sheridan, USA) and feeding tube (size 5 Fr, 1.7 mm; 16 inches (41 cm) inlength, Kendall, USA), and a 1 ml challenge virus, followed by 3 ml ofatmospheric air was administered into nostrils using a syringe.

For oronasal challenge, the challenge virus was administered as a mistusing a nebulizer (NEBULAIR™, DVM Pharmaceuticals, Inc., Miami, Fla.) inapproximately 2 ml volume. The dogs were observed for flu-relatedclinical signs for 14 days post-challenge. The dogs were euthanized atday 14 post challenge, and tissue (lung and trachea) samples werecollected in 10% buffered formalin for histopathological examination.

Results:

All dogs in groups 1 and 2 developed canine influenza clinical signswithin 24 to 48 hours. Each dog had 2 or more of the following clinicalsigns: fever (>103.0° F.; >39.4° C.), cough, serous or mucopurulentocular discharge, serous or mucopurulent nasal discharge, vomiting,diarrhea, depression, weight loss, gagging, hemoptysis, and audiblerales. Lung tissues from 5 of 5 dogs from group 1 and 4 of 5 dogs fromgroup 2 had histopathological lesions which included one or more of thefollowing: diffuse suppurative bronchopneumonia,bronchitis/bronchoiolitis with plugs of neutrophilic exudate in thelumina and marked mononuclear cell aggregation in mucosa andperibronchiolar tissue, mixed exudate within alveoli with large numbersof foamy macrophages, lymphocellular and plasma cellular as well asgranulocytic cell infiltration, and thickening of alveolar septa withproliferation of type II pneumocytes compatible with or pathognomic toan influenza virus infection. The trachea tissue samples were normal.

Conclusion:

An H3N8 canine influenza isolate such as the one used in this study maybe used for inducing canine influenza disease in dogs using one of themethods described in this study or a similar method.

Example 21 Canine Influenza Challenge Model Development

The potential for using a canine influenza virus, H3N8, to develop acanine influenza challenge model in dogs was further investigated in thefollowing study.

Procedure:

Fifteen 17- to 18-week-old mongrels and five 15-week-old beagles wereobtained from commercial suppliers, and were housed in cages in a BSL-2facility. The mongrels were randomly assigned to 3 groups (Groups 1 to3) of 5 dogs each. All beagles were assigned to one group (Group 4) asshown in Table 34:

TABLE 34 Experimental design Group Breed Number of dogs Challenge virusdose 1 Mongrels 5 10^(6.8) TCID₅₀ 2 Mongrels 5 10^(5.8) TCID₅₀ 3Mongrels 5 10^(4.8) TCID₅₀ 4 Beagles 5 10^(6.8) TCID₅₀The dogs were challenged oronasally with a virulent canine influenzavirus, A/Canine/Florida/242/2003 (isolated from lung of a greyhound dogwith canine influenza disease (provided by Dr. Cynda Crawford at theUniversity of Florida)). The challenge virus was administered as a mistusing a nebulizer (NEBULAIR™) in approximately 2 ml volume. The dogswere observed for flu-related clinical signs for 14 days post-challenge.

Results:

Eighty percent (4 of 5) of the dogs in Group 1 and 4, 100% of the dogsin Group 2 and 3, developed canine influenza clinical signs within 48hours. Each dog had one or more of the following clinical signs: fever(>103.0° F.; >39.4° C.), cough, serous or mucopurulent ocular discharge,serous or mucopurulent nasal discharge, vomiting, diarrhea, depression,weight loss, gagging, and rales. The clinical signs observed in beagleswere generally milder and short-course compared to mongrels.

Conclusion:

An H3N8 canine influenza isolate such as the one used in this study maybe used for inducing canine-influenza-like or kennel-cough-like diseasein dogs using method described in this study or a similar method with achallenge dose range from 104.8 to 106.8 TCID50. There were somedifferences in clinical signs observed in mongrels and beagles. Ingeneral, beagles tend to have milder flu-related clinical signs comparedto mongrels.

Example 22 Canine Influenza Vaccine Efficacy Study

The following study was conducted to assess the efficacy of an H3N8equine influenza vaccine in dogs against canine influenza virus.

Procedure:

Seventeen 14-week-old mongrels and ten 8-week-old beagles were obtainedfrom commercial suppliers. The dogs were randomly assigned to 5 groupsas shown in Table 35, and housed in a research facility.

TABLE 35 Experimental design Age at Number of Number of VaccinationGroup Age dogs Treatment doses (weeks) 1 14 weeks 7 Vaccinate 2 14 & 182 14 weeks 5 Vaccinate 1 18 3 14 weeks 5 Control — — 4  8 weeks 5Vaccinate 2  8 & 12 5  8 weeks 5 Control — —

The vaccine used in this study was a HAVLOGEN®-adjuvanted, inactivatedequine influenza virus (A/equine/KY/02) vaccine. To prepare thisvaccine, the virus was inactivated by binary ethylenimine (BEI) using astandard method. Each vaccine dose contained HAVLOGEN® (10% v/v), 6144HA units of the inactivated virus, 0.1% (v/v) of 10% thimerosal, 0.1%(v/v) of phenol red, sufficient NaOH to adjust the pH to from 6.8 to7.2, and sufficient PBS to bring the total dose volume to 1 ml.

The dogs in Groups 1 and 4 were vaccinated with 2 doses of the vaccine.The second dose (i.e., the booster) was administered 4 weeks after thefirst. The dogs in Group 2 were vaccinated with 1 dose at 18weeks-of-age. Blood samples were collected to assess HI titer using astandard protocol (e.g., SAM 124, CVB, USDA, Ames, Iowa) with an H3N8canine influenza isolate on days zero (before vaccination), 7, and 14post first and second vaccinations. Approximately 5 days beforechallenge, the dogs were moved to a BSL-2 facility and housed inindividual cages.

All vaccinates and age-matched control dogs were challenged oronasallywith a virulent canine influenza virus (10^(7.7) TCID50 ofA/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination ofGroups 1 and 4 and first vaccination of Group 2. The challenge virus wasadministered as a mist using a nebulizer (NEBULAIR™) at 2 ml per dog.The dogs were observed for influenza-related clinical signs for 17 dayspost-challenge. Nasal and oropharygeal swabs were collected in tubescontaining 2 ml of virus transport medium for virus isolation from day−1 (i.e., one day before challenge) to day 17 days post-challenge. Alldogs were euthanized at day 17 post-challenge and lung and trachealsamples were collected in 10% buffered formalin for histopathology.Blood samples were collected on days 7 and 14 post challenge for HItiter determination. The clinical sign score assignments used for thepost challenge observation are shown in Table 36.

Results:

All dogs in 2-dose vaccination groups (Group 1 and 4) developed HIantibody titer responses to the canine influenza virus isolate (Table37). Following the challenge, approximately a 4-fold increase in titeron day 14 post challenge in all groups indirectly indicated that alldogs were exposed to the challenge virus. All dogs exhibited one or moreof the following signs of canine influenza: fever (>103.0° F.; >39.4°C.), cough, serous or mucopurulent ocular discharge, serous ormucopurulent nasal discharge, vomiting, diarrhea, depression, weightloss, and dyspnea. Vaccinates had less severe clinical signs, comparedto age-matched controls (Table 38). There was a significant reduction inclinical signs due to the 2-dose vaccination in both 8-week-old(P=0.040) and 14-week-old (P=0.003) dogs (Groups 4 and 1 respectively).In this experiment, one-dose vaccination did not provide a significant(P=0.294) reduction in clinical signs (Group 2)

Virus isolation results are shown in Table 39. Among 14-week-old dogs,canine influenza virus was isolated from swab samples collected from 2of 7 dogs (29%) from the 2-dose vaccine group (Group 1), 3 of 5 dogs(60%) from the 1-dose vaccine group (Group 2), and 5 of 5 dogs (100%)from the control group (Group 3). Among 8-week-old dogs, the virus wasisolated from 1 of 5 dogs (20%) from the 2-dose vaccine group (Group 4),and 4 of 5 dogs (80%) from the control group (Group 5). There was asignificant reduction (P=0.003) in the number of dogs positive forcanine influenza virus in swab samples due to 2-dose vaccination (Groups1 and 4) compared to unvaccinated controls (Groups 3 and 5). Althoughthere was a reduction in the number of dogs (60% vs. 100%) positive forcanine influenza virus in swab samples between 1-dose vaccine group(Group 2) and the control group (Group 3), the difference was notstatistically significant (P=0.222).

Histopathological evaluation of lung and tracheal tissue samples forlesions was conducted to identify lesions compatible with or pathognomicto canine influenza disease. This includes, for example, determinationof whether one or more of the following exist: areas with suppurativebronchopneumonia; peribronchitis/peribronchiolitis with mononuclear cellaggregation (lymphocytes, plasma cells); presence of plugs ofgranulocytic cellular debris in the lumina; hyperplasia of respiratoryepithelium; mixed exudate in the alveoli with large amount ofgranulocytic cells and cell debris; aggregates of (foamy) macrophages,plasma cells, and lymphocytes; and thickening of alveolar septa withproliferation of type II pneumocytes.

Table 40 provides a summary of the extent of lesions in this experimentfor the dogs. Among 14-week-old dogs, the lung lesions were lessextensive and less severe in 5 of 7 dogs in the 2-dose vaccination group(Group 2), and 4 of 5 dogs in the 1-dose vaccination group (Group 1).All controls dogs (Group 3) had severe and extensive lesions suggestiveof no protection. There was no difference in tracheal lesions due to 1-or 2-dose vaccination among 14-week-old dogs. Among 8-week-old dogs,there was no difference in lung lesions between 2-dose vaccinates andcontrol dogs. None of the dogs had any tracheal lesions.

Conclusion:

The results from this study demonstrate that: (1) inactivated H3N8equine influenza virus can induce canine influenza virus cross reactiveHI antibody responses in vaccinated dogs, (2) use of an H3N8 equineinfluenza virus vaccine can reduce the severity of canine influenzavirus disease in dogs, and (3) use of an H3N8 equine influenza virusvaccine can reduce virus excretion in nasal and/or oral secretions.

TABLE 36 Clinical signs and scoring system Clinical signs Score per dayTemp <103.0° F. (<39.4° C.) 0 103.0-103.9° F. (39.4- 2 104.0-104.9° F.(40.0-40.5° C.) 3 >105.0° F. (>40.6° C.) 4 Coughing No coughing 0Occasional 2 Paroxysmal 4 Sneezing No sneezing 0 Occasional 1 Paroxysmal2 Nasal discharge No discharge 0 Serous -slight 1 Serous -copious 1Mucopurulent-slight 2 Mucopurulent-copious 3 Ocular discharge Nodischarge 0 Serous -slight 1 Serous -copious 1 Mucopurulent-slight 2Mucopurulent-copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1Anorexia No 0 Yes 1 Respiratory signs None 0 Rales 3 Dyspnea 4 Gasping 5Mucous expectorate No 0 Yes 2 Vomiting No 0 Yes 1 Fecal abnormalities No0 Yes 1

TABLE 37 Serology - Hemagglutination inhibition titers HI titer Dayspost Group Dog Age Number Days post first vaccination of Groups 1 and 4challenge No ID (week) Treatment of doses 0* 7 14 28** 35 42*** 7 14 1921 14 Vaccinate 2 <10 <10 10 20 40 20 160 320 1 926 14 Vaccinate 2 <10<10 <10 40 40 80 80 >640 1 931 14 Vaccinate 2 <10 <10 <10 10 20 2080 >640 1 955 14 Vaccinate 2 <10 <10 <10 10 40 40 160 320 1 011 14Vaccinate 2 <10 <10 <10 10 20 40 160 320 1 013 14 Vaccinate 2 <10 <10<10 20 40 40 160 320 1 019 14 Vaccinate 2 <10 <10 <10 10 20 40 80 >640 2922 14 Vaccinate 1 <10 <10 <10 <10 <10 <10 >640 >640 2 953 14 Vaccinate1 <10 <10 <10 <10 <10 <10 320 >640 2 015 14 Vaccinate 1 <10 <10 <10 <10<10 <10 320 >640 2 016 14 Vaccinate 1 <10 <10 <10 <10 <10 <10 160 320 2017 14 Vaccinate 1 <10 <10 <10 <10 <10 <10 320 >640 3 923 14 Control N/A<10 <10 <10 <10 <10 <10 40 160 3 012 14 Control N/A <10 <10 <10 <10 <10<10 40 320 3 014 14 Control N/A <10 <10 <10 <10 <10 <10 40 160 3 018 14Control N/A <10 <10 <10 <10 <10 <10 40 160 3 01A 14 Control N/A <10 <10<10 <10 <10 <10 40 160 4 406 8 Vaccinate 2 <10 <10 10 40 80 80 160 >6404 407 8 Vaccinate 2 <10 20 20 40 40 40 320 >640 4 504 8 Vaccinate 2 <10<10 10 20 20 80 160 >640 4 704 8 Vaccinate 2 <10 <10 10 40 80 160160 >640 4 705 8 Vaccinate 2 <10 <10 <10 40 80 160 160 >640 5 404 8Control N/A <10 <10 <10 <10 <10 <10 80 160 5 405 8 Control N/A <10 <10<10 <10 <10 <10 80 80 5 610 8 Control N/A <10 <10 <10 <10 <10 <10 20 405 702 8 Control N/A <10 <10 <10 <10 <10 <10 80 160 5 703 8 Control N/A<10 <10 <10 <10 <10 <10 40 160 *First vaccination - Groups 1 and 4**Second vaccination - Groups 1 and 4; First vaccination - Group 2***Day of challenge

TABLE 38 Analysis of total canine influenza disease clinical scoresNumber of doses Age at first vaccination Average total Group Treatmentof vaccine of Groups 1 and 4 Score per dog P-value* 1 Vaccinate 2 14weeks 8.7 0.003 (Group 1 vs. 3) 2 Vaccinate 1 14 weeks 21.8 0.294 (thesedogs were (Group 2 vs. 3) vaccinated once, when they were 18 weeks old)3 Control — 14 weeks 25.4 — (these dogs were not vaccinated) 4 Vaccinate2  8 weeks 2.0 0.040 (Group 4 vs. 5) 5 Control —  8 weeks 5.4 — (thesedogs were not vaccinated) *Analyzed using a NPARIWAY procedure of SAS ®Version 8.2 (the vaccine groups were compared using the Wilcoxon ranksum test)

TABLE 39 Virus shedding Number of Group Dog Age vaccine Dayspost-challenge No ID (week) Treatment doses −1 0 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 1 921 14 Vaccinate 2 N N N N N N N N N N N N N N N N NN N 1 926 14 Vaccinate 2 N N N N N N N N N N N N N N N N N N N 1 931 14Vaccinate 2 N N N N N N N N N N N N N N N N N N N 1 955 14 Vaccinate 2 NN N N N N N N N N N N N N N N N N N 1 011 14 Vaccinate 2 N N P N N P N NN N N N N N N N N N N 1 013 14 Vaccinate 2 N N N N N P N N N N N N N N NN N N N 1 019 14 Vaccinate 2 N N N N N N N N N N N N N N N N N N N 2 92214 Vaccinate 1 N N N N N N N N N N N N N N N N N N N 2 953 14 Vaccinate1 N N N N N N N N N N N N N N N N N N N 2 015 14 Vaccinate 1 N N N P N PP N N N N N N N N N N N N 2 016 14 Vaccinate 1 N N N P N P P N N N N N NN N N N N N 2 017 14 Vaccinate 1 N N N N P P N N N N N N N N N N N N N 3923 14 Control N/A N N N N N N P N N N N N N N N N N N N 3 012 14Control N/A N N N P N P N N N N N N N N N N N N N 3 014 14 Control N/A NN P N N P P N N N N N N N N N N N N 3 018 14 Control N/A N N N P P P N NN N N N N N N N N N N 3 01A 14 Control N/A N N N P P P P N N N N N N N NN N N N 4 406 8 Vaccinate 2 N N N N N N N N N N N N N N N N N N N 4 4078 Vaccinate 2 N N N N N N N N N N N N N N N N N N N 4 504 8 Vaccinate 2N N N P N N N N N N N N N N N N N N N 4 704 8 Vaccinate 2 N N N N N N NN N N N N N N N N N N N 4 705 8 Vaccinate 2 N N N N N N N N N N N N N NN N N N N 5 404 8 Control N/A N N P P N N P N N N N N N N N N N N N 5405 8 Control N/A N N N P N N P N N N N N N N N N N N N 5 610 8 ControlN/A N N N N N N N N N N N N N N N N N N N 5 702 8 Control N/A N N N P NN N N N N N N N N N N N N N 5 703 8 Control N/A N N N N N N P N N N N NN N N N N N N

TABLE 40 Histopathological evaluation of tissue samples Microscopiclesion Group Dog Age Number (Histopathology) No ID (week) Treatment ofdoses Lungs Trachea 1 921 14 Vaccinate 2 +/− − 1 926 14 Vaccinate 2 −+/− 1 931 14 Vaccinate 2 − − 1 955 14 Vaccinate 2 +/− − 1 011 14Vaccinate 2 +/− − 1 013 14 Vaccinate 2 +/− +/− 1 019 14 Vaccinate 2 +/−+/− 2 922 14 Vaccinate 1 +/− − 2 953 14 Vaccinate 1 +/− +/− 2 015 14Vaccinate 1 +/− + 2 016 14 Vaccinate 1 − − 2 017 14 Vaccinate 1 +/− +/−3 923 14 Control N/A + +/− 3 012 14 Control N/A + − 3 014 14 ControlN/A + − 3 018 14 Control N/A + − 3 01A 14 Control N/A + +/− 4 406 8Vaccinate 2 +/− − 4 407 8 Vaccinate 2 − − 4 504 8 Vaccinate 2 +/− − 4704 8 Vaccinate 2 − − 4 705 8 Vaccinate 2 − − 5 404 8 Control N/A − − 5405 8 Control N/A − − 5 610 8 Control N/A +/− − 5 702 8 Control N/A +/−− 5 703 8 Control N/A − − “+” Severe lesion consistent or pathognomic toan influenza infection “+/−” Mild lesion (inconclusive) “−” Normal

Example 23 Canine Influenza Vaccine Efficacy Study

The following study was conducted to determine the efficacy of amultivalent H3N8 equine influenza vaccine against canine influenza virusin dogs.

Procedure:

Seventeen 15-week-old beagles were obtained from a commercial supplier.The dogs were randomly assigned to 3 groups as shown in Table 41, andhoused in a research facility.

TABLE 41 Experimental design Age at Number of Vaccination Group Numberof dogs Treatment doses (weeks) 1 7 Vaccinate 2 15 & 19 2 5 Vaccinate 119 3 5 Control — —

The vaccine used in this study was a HAVLOGEN® adjuvanted, inactivatedequine influenza (A/equine/KY/02, A/equine/KY/93, and A/equine/NM/2/93)vaccine. To prepare this vaccine, the viruses were inactivated by binaryethylenimine (BEI) using a standard method. Each vaccine dose containedHAVLOGEN® (10% v/v), 2048 HA units of each of the inactivated virus,0.1% (v/v) of 10% thimerosal, 0.1% (v/v) of phenol red, sufficient NaOHto adjust the pH to 6.8 to 7.2, and sufficient PBS to bring the totaldose volume to 1 ml.

The dogs in Group 1 were vaccinated with 2 doses of the vaccine. Thesecond (i.e., booster) dose was administered 4 weeks after the firstdose. The dogs in Group 2 were vaccinated with 1 dose of vaccine at 19weeks-of-age. Blood samples were collected to assess HI titer using astandard protocol with an H3N8 canine influenza isolate on days zero(before vaccination), 7, and 14 post first and second vaccinations.Seven days before challenge, the dogs were moved to a BSL-2 facility andhoused in individual cages.

All vaccinates and age-matched control dogs were challenged oronasallywith a virulent canine influenza virus (10^(7.3) TCID50 ofA/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination ofGroup 1 and first vaccination of Group 2. The challenge virus wasadministered as a mist using a nebulizer (NEBULAIR™) at 2 ml per dog.The dogs were observed for influenza-related clinical signs for 14 dayspost challenge. All dogs were euthanized at day 14 post-challenge, andlung and trachea samples were collected in 10% buffered formalin forhistopathology. Blood samples were collected on days 7 and 14 postchallenge for HI titer determination. The clinical sign scoreassignments used for the post challenge observation are shown in Table42.

Results:

All vaccinated dogs developed HI antibody titer responses to the canineinfluenza virus isolate (Table 43). Following the challenge,approximately a 4 fold increase in HI titer on day 14 post challengecompared to the pre-challenge HI titer in all groups indirectly indicatethat all dogs were exposed to the challenge virus. All dogs exhibitedsigns canine influenza disease with each dog demonstrating one or moreof the following clinical signs: fever (>103.0° F.; >39.4° C.), cough,serous or mucopurulent ocular discharge, serous or mucopurulent nasaldischarge, vomiting, diarrhea, depression, weight loss, and dyspnea.Vaccinates had less severe clinical signs, compared to age-matchedcontrols (Table 44). There was a significant (P=0.028) reduction inclinical signs due to the 2-dose vaccination in dogs (Group 1). One dosevaccination did not provide a significant (P=0.068) reduction inclinical signs (Group 2).

As in Example 22, histopathological evaluation of lung and trachealtissue samples for lesions was conducted to identify lesions compatiblewith or pathognomic to canine influenza disease. Table 45 provides asummary of the extent of lesions in this experiment for the dogs. Among15-week-old dogs, vaccination of dogs with either 1 dose or 2 dosesprevented the lung lesions in all dogs. Four of 5 control dogs (80%) hadsevere suppurative bronchopneumonia consistent with an influenzadisease. One of 7 dogs from the 2-dose vaccine group (Group 1) and 1 of5 dogs from the control group (Group 3) had mild trachea lesionssuggestive of tracheitis which could be attributed to influenza disease.

Conclusion:

The results from this study demonstrate that 1) inactivated H3N8 equineinfluenza virus can induce canine influenza virus cross reactive HIantibody responses in vaccinated dogs, and 2) Use of a H3N8 equineinfluenza virus vaccine can reduce the severity of canine influenzavirus disease in dogs.

TABLE 42 Clinical signs and scoring system Clinical signs Score per dayTemp <103.0° F. (<39.4° C.) 0 103.0-103.9° F. (39.4- 2 104.0-104.9° F.(40.0- 3 >105.0° F. (>40.6° C.) 4 Coughing No coughing 0 Occasional 2Paroxysmal 4 Sneezing No sneezing 0 Occasional 1 Paroxysmal 2 Nasaldischarge No discharge 0 Serous -slight 1 Serous -copious 1Mucopurulent-slight 2 Mucopurulent-copious 3 Ocular discharge Nodischarge 0 Serous -slight 1 Serous -copious 1 Mucopurulent-slight 2Mucopurulent-copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1Anorexia No 0 Yes 1 Respiratory signs None 0 Rales 3 Dyspnea 4 Gasping 5Mucous expectorate No 0 Yes 2 Vomiting No 0 Yes 1 Fecal abnormalities No0 Yes 1

TABLE 43 Serology - Hemagglutination inhibition titers HI titer Dayspost Group Dog Number Days post first vaccination of Group 1 challengeNo ID Treatment of doses 0* 7 14 28** 35 42*** 7 14 1 ALK Vaccinate 2<10 <10 20 20 80 40 160 320 1 AMF Vaccinate 2 <10 <10 10 20 20 40 160320 1 AKY Vaccinate 2 <10 20 20 20 40 40 160 80 1 ALC Vaccinate 2 <10 1010 10 40 40 160 160 1 ALL Vaccinate 2 <10 <10 10 10 40 20 160 320 1 ALMVaccinate 2 <10 <10 10 20 40 40 80 160 1 AMU Vaccinate 2 <10 20 40 40 4040 40 160 2 ALA Vaccinate 1 <10 <10 <10 <10 <10 10 320 160 2 AMAVaccinate 1 <10 <10 <10 <10 <10 20 >640 80 2 APD Vaccinate 1 <10 <10 <10<10 <10 10 >640 320 2 APG Vaccinate 1 <10 <10 <10 <10 <10 10 320 80 2APT Vaccinate 1 <10 <10 <10 <10 <10 10 320 320 3 ALT Control N/A <10 <10<10 <10 <10 <10 40 160 3 AMS Control N/A <10 <10 <10 <10 <10 <10 80 1603 AKX Control N/A <10 <10 <10 <10 <10 <10 20 80 3 ALX Control N/A <10<10 <10 <10 <10 <10 80 80 3 AMI Control N/A <10 <10 <10 <10 <10 <10 4080 *First vaccination - Group 1 **Second vaccination - Group 1; Firstvaccination - Group 2 ***Day of challenge

TABLE 44 Analysis of total canine influenza disease clinical scoresNumber Age at first vaccination Average total Group Treatment of dosesof Group 1 Score per dog P-value* 1 Vaccinate 2 15 weeks 6.3 0.028(Group 1 vs. 3) 2 Vaccinate 1 15 weeks 14.2 0.068 (these dogs were(Group 2 vs. 3) vaccinated once, when they were 19 weeks old) 3 Control— 15 weeks 24.4 — (these dogs were not vaccinated) *Analyzed using aNPARIWAY procedure of SAS ® Version 8.2 (the vaccine groups werecompared using the Wilcoxon rank sum test)

TABLE 45 Histopathological evaluation of tissue samples Microscopiclesion Group Number of (Histopathology) No Dog ID Treatment doses LungTrachea 1 ALK Vaccinate 2 − +/− 1 AMF Vaccinate 2 − − 1 AKY Vaccinate 2− − 1 ALC Vaccinate 2 − − 1 ALL Vaccinate 2 − − 1 ALM Vaccinate 2 − − 1AMU Vaccinate 2 − − 2 ALA Vaccinate 1 − − 2 AMA Vaccinate 1 − − 2 APDVaccinate 1 − − 2 APG Vaccinate 1 − − 2 APT Vaccinate 1 − − 3 ALTControl N/A +/− − 3 AMS Control N/A + − 3 AKX Control N/A + − 3 ALXControl N/A + +/− 3 AMI Control N/A − − “+” Severe lesion consistent orpathognomic to an influenza infection “+/−” Mild lesions (inconclusive)“−” Normal

Example 24 Canine Influenza Vaccine Efficacy Study

The following study was conducted to determine: (1) the efficacy ofmonovalent versus multivalent H3N8 equine influenza vaccines againstcanine influenza virus in dogs, and (2) the effect of route ofadministration on vaccine efficacy.

Procedure:

Thirty 10-week old mongrels were obtained from a commercial supplier.The dogs were randomly assigned to 6 groups as shown in Table 46, andhoused in a research facility.

TABLE 46 Experimental design Age at Number of Route of Number ofVaccination Group dogs Treatment vaccination doses (weeks) 1 5 VAX-1 IN2 10 & 14 2 5 VAX-2 SQ 2 10 & 14 3 5 VAX-2 IN 2 10 & 14 4 5 VAX-3 SQ 210 & 14 5 5 VAX-3 IN 2 10 & 14 6 5 Control — — —

Three types of vaccines (VAX-1, VAX-2, and VAX-3) were used. The VAX-1was a HAVLOGEN®-adjuvanted, inactivated equine influenza virus(A/equine/KY/02) monovalent vaccine, and each dose contained HAVLOGEN®(10% v/v), 6144 HA units of the inactivated virus, 0.1% (v/v) of 10%thimerosal, 0.1% (v/v) of phenol red, sufficient NaOH to adjust the pHto 6.8 to 7.2, and sufficient PBS to bring the total dose volume to 1ml. The VAX-2 was a HAVLOGEN®-adjuvanted, inactivated equine influenzavirus (A/equine/KY/02) monovalent vaccine, and each dose of vaccinecontained HAVLOGEN® (10% v/v), 4096 HA units of the inactivated virus,0.1% (v/v) of 10% thimerosal, 0.1% (v/v) of phenol red, sufficient NaOHto adjust the pH to 6.8 to 7.2, and sufficient PBS to bring the totaldose volume to 1 ml. The VAX-3 was a HAVLOGEN®-adjuvanted, inactivatedequine influenza (A/equine/KY/02, A/equine/KY/93, and A/equine/NM/2/93)multivalent vaccine, and contained HAVLOGEN® (10% v/v), 2048 HA units ofinactivated virus per strain, 0.1% (v/v) of 10% thimerosal, 0.1% (v/v)of phenol red, sufficient NaOH to adjust the pH to 6.8 to 7.2, andsufficient PBS to bring the total dose volume to 1 ml. All influenzaviruses used for the vaccine formulation were inactivated by binaryethylenimine (BEI) using a standard method.

The vaccines and routes of administration for each group are describedin Table 46. All dogs in the vaccinated groups were vaccinated eithervia the intranasal (IN) or the subcutaneous (SQ) route, and each dogreceived 2 doses. The second (i.e., booster) dose was administered 4weeks after the first dose. Blood samples were collected to assess HItiter using a standard protocol with an H3N8 canine influenza isolate ondays zero (before vaccination), 7, and 14 post first and secondvaccinations. Seven days before challenge, the dogs were moved to aBSL-2 facility and housed in individual cages.

All vaccinates and age-matched control dogs were challenged oronasallywith a virulent canine influenza virus (10^(7.4) TCID50 ofA/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination.The challenge virus was administered as a mist using a nebulizer(NEBULAIR™) in a 2 ml volume per day. The dogs were observed forinfluenza-related clinical signs for 14 days post-challenge. Bloodsamples were collected on days 7 and 14 post challenge for HI titerdetermination. All dogs were euthanized at day 14 post-challenge, andlung and trachea samples were collected in 10% buffered formalin forhistopathology. The clinical sign score assignments used for the postchallenge observation are shown in Table 47.

Results:

All dogs vaccinated via the SQ route developed HI antibody titerresponses to the canine influenza virus isolate, regardless of thevaccine type (Table 48). None of the dogs from the IN vaccination groups(i.e., Groups 1, 3, and 5) developed HI antibody titer responses to thecanine influenza virus isolate, regardless of the vaccine type, duringthe post vaccination period. There was, however, a 4-fold increase intiter by day 14 post challenge in all dogs indirectly, indicating thatall dogs were exposed to the challenge virus (Table 47).

All dogs exhibited one or more of the following clinical signs of canineinfluenza: fever (>103.0° F.; >39.4° C.), cough, serous or mucopurulentocular discharge, serous or mucopurulent nasal discharge, vomiting,diarrhea, depression, weight loss, and dyspnea. Vaccinates had lesssevere clinical signs, compared to age-matched controls (Table 49).There was a significant reduction in clinical signs in dogs vaccinatedwith VAX-3 via the SQ route (Group 4). In this experiment, INadministration of either VAX-1, VAX-2, or VAX-3 did not provide asignificant reduction in clinical signs of canine influenza virus.

As in Examples 22 and 23, histopathological evaluation of lung andtracheal tissue samples for lesions was conducted to identify lesionscompatible with or pathognomic to canine influenza disease. Table 50provides a summary of the extent of lesions in this experiment for thedogs. Five of 5 control dogs (Group 6) had lung lesions consistence withan influenza infection. Two of 5 dogs vaccinated with VAX-2 via the SCroute (Group 2) and 3 of 5 dogs vaccinated with VAX-3 via the SC route(Group 4) were free of any influenza-related lung lesions. All the dogsthat received the vaccine via the intranasal route, irrespective of thevaccine type, had severe lung lesions consistent with an influenzainfection. The trachea lesions observed in this study were very mild.

Conclusion:

The results from this study demonstrate that: (1) inactivated H3N8equine influenza virus can induce canine influenza virus cross reactiveHI antibody responses in dogs vaccinated via the SQ route, (2)intranasal administration of either monovalent (VAX-1 and VAX-2) ormultivalent vaccine (VAX-3) was not efficacious in dogs, and (3)subcutaneous administration of multivalent vaccine (VAX-3) provided asignificant (P=0.016) reduction in severity of canine influenza virusdisease in dogs.

TABLE 47 Clinical signs and scoring system Clinical signs Score per dayTemp <103.0° F. (<39.4° C.) 0 103.0-103.9° F. (39.4- 2 104.0-104.9° F.(40.0- 3 >105.0° F. (>40.6° C.) 4 Coughing No coughing 0 Occasional 2Paroxysmal 4 Sneezing No sneezing 0 Occasional 1 Paroxysmal 2 Nasaldischarge No discharge 0 Serous -slight 1 Serous -copious 1Mucopurulent-slight 2 Mucopurulent-copious 3 Ocular discharge Nodischarge 0 Serous -slight 1 Serous -copious 1 Mucopurulent-slight 2Mucopurulent-copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1Anorexia No 0 Yes 1 Respiratory signs None 0 Rales 3 Dyspnea 4 Gasping 5Mucous expectorate No 0 Yes 2 Vomiting No 0 Yes 1 Fecal abnormalities No0 Yes 1

TABLE 48 Serology - Hemagglutination inhibition titers HI titer Dayspost Group Dog Route of Number Days post vaccination challenge No IDTreatment vaccination of doses 0* 7 14 28** 35 42*** 7 14 1 248Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80 40 1 501 Vaccinate IN 2 <10 10<10 <10 <10 <10 160 160 1 502 Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80160 1 469 Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80 160 1 46A VaccinateIN 2 <10 <10 <10 <10 <10 <10 80 80 2 232 Vaccinate SQ 2 <10 <10 <10 2020 40 320 640 2 511 Vaccinate SQ 2 <10 10 10 20 20 20 160 640 2 514Vaccinate SQ 2 <10 <10 40 40 80 40 160 320 2 461 Vaccinate SQ 2 <10 1010 20 20 20 >640 >640 2 463 Vaccinate SQ 2 <10 10 40 80 80 40 80 320 3246 Vaccinate IN 2 <10 10 <10 <10 <10 <10 40 40 3 505 Vaccinate IN 2 <10<10 <10 <10 <10 <10 80 80 3 506 Vaccinate IN 2 <10 <10 <10 <10 <10 <1080 160 3 464 Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80 80 3 465Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80 160 4 23B Vaccinate SQ 2 <1010 10 40 40 20 160 160 4 247 Vaccinate SQ 2 <10 <10 <10 20 20 20 160 3204 508 Vaccinate SQ 2 <10 10 40 40 80 80 320 320 4 512 Vaccinate SQ 2 <10<10 20 20 80 80 320 160 4 516 Vaccinate SQ 2 <10 10 10 20 80 80 160 >6405 503 Vaccinate IN 2 <10 10 <10 <10 <10 <10 80 160 5 513 Vaccinate IN 2<10 <10 <10 <10 <10 <10 80 80 5 462 Vaccinate IN 2 <10 <10 <10 <10 <10<10 80 320 5 466 Vaccinate IN 2 <10 <10 <10 <10 <10 <10 80 80 5 46BVaccinate IN 2 <10 <10 <10 <10 <10 <10 80 160 6 236 Control — 2 <10 <10<10 <10 <10 <10 80 160 6 504 Control — 2 <10 <10 <10 <10 <10 <10 160 1606 507 Control — 2 <10 <10 <10 <10 <10 <10 80 160 6 515 Control — 2 <10<10 <10 <10 <10 <10 80 160 6 468 Control — 2 <10 <10 <10 <10 <10 <10 80160 *First vaccination **Second vaccination ***Day of challenge

TABLE 49 Analysis of total canine influenza disease clinical scoresRoute of Average total Group Treatment vaccination Score per dogP-value* 1 VAX-1 IN 35.2 0.500 (Group 1 vs. 6) 2 VAX-2 SQ 31.0 0.345(Group 2 vs. 6) 3 VAX-2 IN 39.4 0.631 (Group 3 vs. 6) 4 VAX-3 SQ 13.00.016 (Group 4 vs. 6) 5 VAX-3 IN 42.6 0.790 (Group 4 vs. 6) 6 Control —36.8 — *Analyzed using a NPARIWAY procedure of SAS ® Version 8.2 (thevaccine groups were compared using the Wilcoxon rank sum test)

TABLE 50 Histopathological evaluation of tissue samples Microscopiclesion Group Dog Route of Number (Histopathology) No ID Treatmentvaccination of doses Lung Trachea 1 248 Vaccinate IN 2 + − 1 501Vaccinate IN 2 + − 1 502 Vaccinate IN 2 + − 1 469 Vaccinate IN 2 + + 146A Vaccinate IN 2 + + 2 232 Vaccinate SQ 2 + − 2 511 Vaccinate SQ 2 + −2 514 Vaccinate SQ 2 − − 2 461 Vaccinate SQ 2 + − 2 463 Vaccinate SQ 2 −− 3 246 Vaccinate IN 2 + − 3 505 Vaccinate IN 2 + − 3 506 Vaccinate IN2 + + 3 464 Vaccinate IN 2 + − 3 465 Vaccinate IN 2 + + 4 23B VaccinateSQ 2 − − 4 247 Vaccinate SQ 2 +/− − 4 508 Vaccinate SQ 2 − − 4 512Vaccinate SQ 2 − +/− 4 516 Vaccinate SQ 2 + + 5 503 Vaccinate IN 2 + +/−5 513 Vaccinate IN 2 + + 5 462 Vaccinate IN 2 + +/− 5 466 Vaccinate IN2 + + 5 46B Vaccinate IN 2 + − 6 236 Control — 2 + − 6 504 Control —2 + + 6 507 Control — 2 + + 6 515 Control — 2 + +/− 6 468 Control —2 + + “+” Severe lesion consistent or pathognomic to an influenzainfection “+/−” Mild lesion (inconclusive) “−” Normal

Example 25 Canine Influenza Vaccine Efficacy Study

Canine influenza disease is caused by an H3N8 influenza virus (CIV). CIVis very closely related to equine H3N8 viruses (Crawford et al., 2005)and infects all exposed dogs. Approximately 80% of exposed dogs developclinical signs. In the following study the efficacy of an inactivatedH3N8 equine influenza virus vaccine and a canine influenza virus vaccinewere determined.

Procedure:

Thirty-five beagles and five mongrels were used in this study. Beagleswere randomly assigned to three groups (Table 51). All mongrels wereassigned to control group (Group 3). All dogs were fed with a standardgrowth diet and water was available as libitum.

TABLE 51 Experimental design Age at Vaccination Number of vaccinationGroup Treatment route dogs (weeks) Challenge 1 VAX-1 IM 15 8 & 12 Yes 2VAX-2 SC 5 8 & 12 Yes 3 Control N/A 20 N/A YesThe dogs in Groups 1 and 2 were vaccinated with either VAX-1 or VAX-2(Table 51). VAX-1 was a HAVLOGEN® adjuvanted, inactivated equineinfluenza virus (A/equine/KY/02) vaccine. For vaccine preparation, thevaccine virus was inactivated by binary ethylenimine (BEI) using astandard method. Each dose of vaccine contained HAVLOGEN® (10% v/v),6144 HA units of the inactivated virus, 0.1% (v/v) of 10% thimerosal,0.1% (v/v) of phenol red and sufficient PBS to bring the total dosevolume to 1 ml and sufficient NaOH to adjust the pH to 6.8 to 7.2.

VAX-2 was an inactivated, CARBIGEN™ adjuvanted, canine influenza antigenvaccine (A/canine/Fl/43/2004). The A/canine/Fl/43/2004 was inactivatedby binary ethylenimine (“BEI”) using a standard method. Each dose of thevaccine contained 5% by mass CARBIGEN™, approximately 1280 HA units ofthe inactivated virus, sufficient PBS to bring the total volume of thedose to 1 ml, and sufficient NaOH to adjust the pH to between 7.2 and7.4. Serum samples were collected from all dogs on the day of first andsecond vaccination, days 7 and 14 post first and second vaccinations,and at pre-challenge to determine the HI titers using an H3N8 equineinfluenza virus standard protocol (SAM 124, CVB, USDA, Ames, Iowa).Seven days before challenge, the dogs were moved to a ABSL-2 facilityand housed in individual cages.

All vaccinates and age-matched control dogs were challenged oronasallywith virulent canine influenza virus (10^(7.2) TCID50 ofA/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination.The challenge virus was administered as a mist (2 ml/dog) using anebulizer (NEBULAIR™). The dogs were observed for influenza-relatedclinical signs for 14 days post-challenge. Nasal and oropharyngeal swabswere collected daily in tubes containing 2 ml of virus transport mediumfor virus isolation from day −1 (i.e., one day before challenge) throughday 14 post-challenge. Blood samples were collected on days 7 and 14post challenge for HI titer determination. The clinical sign scoreassignments used for post challenge observation are shown in Table 52.

Results:

All vaccinated dogs (Groups 1 and 2) developed HI antibody titerresponses to the canine influenza virus isolate (Table 53). All dogsexhibited one or more of the following signs of canine influenza: fever(>103.0° F.; >39.4° C.), cough, serous or mucopurulent ocular discharge,serous or mucopurulent nasal discharge, vomiting, diarrhea, depression,and anorexia. Vaccinates had less severe clinical signs, compared toage-matched controls (Table 54). There was a significant (P<0.001)reduction in clinical signs in dogs vaccinated with either VAX-1(Group 1) or VAX-2 (Group 2).

Virus isolation results are shown in Tables 55 and 56. Following avirulent canine influenza virus challenge, the canine influenza viruswas isolated from 5 of 15 (33%) dogs from Group 1 (VAX-1), 0 of 5 (0%)dogs from Group 2 (VAX-2) and 17 of 20 (85%) controls (Group 3). Bothinactivated equine influenza vaccine (VAX-1) and canine influenza virus(VAX-2) vaccinates demonstrated a significant (P=0.004) reduction invirus shedding in nasal or oral secretions or both (Table 55) comparedto controls.

Conclusion:

The results from this study demonstrate that: (1) inactivated H3N8equine influenza virus and canine influenza virus vaccines can inducecanine influenza virus reactive HI antibody responses in vaccinateddogs, (2) use of an H3N8 equine influenza virus or canine influenzavirus vaccine can reduce the severity of canine influenza virus diseasein dogs, and (3) use of an H3N8 equine influenza virus or canineinfluenza virus vaccine can reduce virus excretion in nasal and/or oralsecretions.

TABLE 52 Clinical signs and scoring system Clinical signs Score per dayTemp <103.0° F. (<39.4° C.) 0 103.0-103.9° F. (39.4- 2 104.0-104.9° F.(40.0-40.5° C.) 3 >105.0° F. (>40.6° C.) 4 Coughing No coughing 0Occasional 2 Paroxysmal 4 Sneezing No sneezing 0 Occasional 1 Paroxysmal2 Nasal discharge No discharge 0 Serous -slight 1 Serous -copious 1Mucopurulent-slight 2 Mucopurulent-copious 3 Ocular discharge Nodischarge 0 Serous -slight 1 Serous -copious 1 Mucopurulent-slight 2Mucopurulent-copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1Anorexia No 0 Yes 1 Respiratory signs None 0 Rales 3 Dyspnea 4 Gasping 5Mucous expectorate No 0 Yes 2 Vomiting No 0 Yes 1 Fecal abnormalities No0 Yes 1

TABLE 53 Serology - Hemagglutination inhibition titers HI titer GroupDog Vaccination Days post vaccination Days post challenge No IDTreatment route 0* 7 14 28** 35 42*** 7 14 1 AYS Vaccinate IM <10 <10<10 20 40 40 80 ≧640 1 AZV Vaccinate IM <10 <10 <10 20 40 40 160 ≧640 1BAD Vaccinate IM <10 <10 <10 40 40 80 80 320 1 BAE Vaccinate IM <10 <1010 20 20 20 40 320 1 BAH Vaccinate IM <10 <10 10 10 40 40 160 ≧640 1 BAJVaccinate IM <10 <10 10 20 80 80 40 320 1 BAN Vaccinate IM <10 10 10 2040 40 40 320 1 BBN Vaccinate IM <10 10 10 20 80 80 40 320 1 BBTVaccinate IM <10 <10 <10 20 40 40 40 160 1 BBY Vaccinate IM <10 <10 <1020 80 80 160 ≧640 1 BCS Vaccinate IM <10 10 40 40 160 160 160 160 1 BCZVaccinate IM <10 10 10 20 80 40 160 160 1 BDP Vaccinate IM <10 <10 <1020 40 40 80 ≧640 1 BEE Vaccinate IM <10 10 20 40 80 80 160 320 1 BEYVaccinate IM <10 <10 10 10 40 40 160 160 2 AZH Vaccinate SC <10 <10 1020 80 80 160 160 2 AZT Vaccinate SC <10 <10 10 10 40 80 320 ≧640 2 BBCVaccinate SC <10 <10 20 40 160 160 80 160 2 BCM Vaccinate SC <10 <10 1020 80 40 80 160 2 BEB Vaccinate SC <10 <10 <10 10 20 40 80 160 3 AYTControl N/A <10 <10 <10 <10 <10 <10 40 320 3 AZJ Control N/A <10 <10 <10<10 <10 <10 20 160 3 AZL Control N/A <10 <10 <10 <10 <10 <10 40 160 3AZN Control N/A <10 <10 <10 <10 <10 <10 160 160 3 BAB Control N/A <10<10 <10 <10 <10 <10 40 320 3 BED Control N/A <10 <10 <10 <10 <10 <10 320≧640 3 BBU Control N/A <10 <10 <10 <10 <10 <10 160 160 3 BBZ Control N/A<10 <10 <10 <10 <10 <10 20 160 3 BCC Control N/A <10 <10 <10 <10 <10 <1040 320 3 BCD Control N/A <10 <10 <10 <10 <10 <10 80 ≧640 3 BCG ControlN/A <10 <10 <10 <10 <10 <10 40 ≧640 3 BCI Control N/A <10 <10 <10 <10<10 <10 20 320 3 BCL Control N/A <10 <10 <10 <10 <10 <10 80 ≧640 3 BCVControl N/A <10 <10 <10 <10 <10 <10 40 320 3 BDU Control N/A <10 <10 <10<10 <10 <10 80 ≧640 3 MFI Control N/A NT NT NT NT <10 <10 80 320 3 MFJControl N/A NT NT NT NT <10 <10 40 320 3 MFK Control N/A NT NT NT NT <10<10 80 320 3 MFR Control N/A NT NT NT NT <10 <10 80 320 3 MFS ControlN/A NT NT NT NT <10 <10 160 ≧640 *First vaccination **Second vaccination***Day of challenge

TABLE 54 Analysis of total canine influenza disease clinical scoresAverage total Group Treatment Score per dog P-value* 1 VAX-1 9.1 <0.001(Group 1 vs. 3) 2 VAX-2 5.4 <0.001 (Group 2 vs. 3) 3 Control 24.1 —*Analyzed using a NPARIWAY procedure of SAS ® Version 9.1 (the vaccinegroups were compared using the GLM procedure)

TABLE 55 Post-challenge virus shedding Percent dogs Group Treatmentexcreted the virus P-value* 1 VAX-1 33% (5/15) 0.004 (Group 1 vs. 3) 2VAX-2 0% (0/5) 0.004 (Group 2 vs. 3) 3 Control  85% (17/20) — *Analyzedusing a FREQ procedure of SAS ® (Version 9.1) and P-value associatedwith Fisher's exact test

TABLE 56 Serology - Hemagglutination inhibition titers Group DogVaccination Days post-challenge No ID Treatment route −1 0 1 2 3 4 5 6 78 9 10 11 12 13 14 1 AYS Vaccinate IM N N N N N N N N N N N N N N N N 1AZV Vaccinate IM N N N P N N N N N N N N N N N N 1 BAD Vaccinate IM N NN N N N N N N N N N N N N N 1 BAE Vaccinate IM N N N P P N N N N N N N NN N N 1 BAH Vaccinate IM N N N N N N N N N N N N N N N N 1 BAJ VaccinateIM N N N P N N N N N N N N N N N N 1 BAN Vaccinate IM N N N N N N N N NN N N N N N N 1 BBN Vaccinate IM N N N N N N N N N N N N N N N N 1 BBTVaccinate IM N N N N N N N N N N N N N N N N 1 BBY Vaccinate IM N N N NN N N N N N N N N N N N 1 BCS Vaccinate IM N N N N N N N N N N N N N N NN 1 BCZ Vaccinate IM N N N N N N N N N N N N N N N N 1 BDP Vaccinate IMN N N P N N N N N N N N N N N N 1 BEE Vaccinate IM N N N N N N N N N N NN N N N N 1 BEY Vaccinate IM N N N P N N N N N N N N N N N N 2 AZHVaccinate SC N N N N N N N N N N N N N N N N 2 AZT Vaccinate SC N N N NN N N N N N N N N N N N 2 BBC Vaccinate SC N N N N N N N N N N N N N N NN 2 BCM Vaccinate SC N N N N N N N N N N N N N N N N 2 BEB Vaccinate SCN N N N N N N N N N N N N N N N 3 AYT Control N/A N N N N N N N N N N NN N N N N 3 AZJ Control N/A N N N N N P P N N N N N N N N N 3 AZLControl N/A N N N N N N P N N N N N N N N N 3 AZN Control N/A N N N P NN N N N N N N N N N N 3 BAB Control N/A N N N P N N N N N N N N N N N N3 BBD Control N/A N N N N N N N N N N N N N N N N 3 BBU Control N/A N NN N N N P N N N N N N N N N 3 BBZ Control N/A N N N P N N P N N N N N NN N N 3 BCC Control N/A N N N P N P N N N N N N N N N N 3 BCD ControlN/A N N N N N P P N N N N N N N N N 3 BCG Control N/A N N N P N P N N NN N N N N N N 3 BCI Control N/A N N N P P P N N N N N N N N N N 3 BCLControl N/A N N N P P N P N N N N N N N N N 3 BCV Control N/A N N N P PN N N N N N N N N N N 3 BDU Control N/A N N N P P N N N N N N N N N N N3 MFI Control N/A N N N P P P P N N N N N N N N N 3 MFJ Control N/A N NN P N N N N N N N N N N N N 3 MFK Control N/A N N N P N N N N N N N N NN N N 3 MFR Control N/A N N N N N N N N N N N N N N N N 3 MFS ControlN/A N N N P P P N N N N N N N N N N N—No virus isolated from oral ornasal swabs P—Virus isolated from nasal or oral or nasal and oral swabs.

TABLE 57 Hemagglutinin, neuraminidase and nucleoprotein gene amino acidsequence similarities among influenza viruses Gene (Canine/ Amino acidsequence Gene of influenza virus Florida/43/2004) similarity used forcomparison Hemagglutinin 88 equine/Algiers/72 HA 90 equine/Saopaulo/6/69 HA 91 equine/Miami/1/63 HA 93 equine/Newmarket/79 HA 94equine/Kentucky/1/81 HA 95 Equi-2/Ludhiana/87 HA 96 Equine/Alaska/1/91HA 97 equine/Tennessee/5/86 HA 98 equine/Kentucky/5/02 HA 99equine/Ohio/1/2003 HA 99 canine/Florida/242/2003 Neuraminidase 88Eq/Algiers/72 NA 90 equine/Sao Paulo/6/69 NA 91 equine/Miami/1/63 NA 93equine/Newmarket/79 NA 94 equine/Kentucky/1/81 NA 95 Equi-2/Ludhiana/87NA 96 equine/Santiago/85 NA 97 equine/Tennessee/5/86 NA 98equine/Kentucky/5/2002 NA 99 equine/Ohio/1/2003 NA 99canine/Florida/242/2003 Nucleoprotein (“NP”) 94 equi/Miami/1/63 NP 97equine/Kentucky/1/81 NP 99 equine/Kentucky/5/02 NP 99 equine/Ohio/1/2003NP 99 canine/Florida/242/2003

The words “comprise,” “comprises,” and “comprising” in this patent(including the claims) are to be interpreted inclusively rather thanexclusively. This interpretation is intended to be the same as theinterpretation that these words are given under United States patentlaw.

The above detailed description of preferred embodiments is intended onlyto acquaint others skilled in the art with the invention, itsprinciples, and its practical application so that others skilled in theart may adapt and apply the invention in its numerous forms, as they maybe best suited to the requirements of a particular use. This invention,therefore, is not limited to the above embodiments, and may be variouslymodified.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

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1. (canceled)
 2. A reassortant virus comprising at least onepolypeptide, gene, genomic segment, or a polynucleotide of an influenzavirus that can infect a canid animal.
 3. The reassortant virus accordingto claim 2, wherein said reassortant virus is formulated in apharmaceutically acceptable carrier or diluent.
 4. An isolated virusthat comprises one or more heterologous polypeptides or polynucleotides,wherein said one or more polypeptides comprises a canine influenza virusprotein selected from HA, NA, NP, MA, PB1, PB2, PA, or NS, or afunctional or immunogenic fragment thereof, or wherein said one or morepolynucleotides encode a canine influenza virus protein selected fromHA, NA, NP, MA, PB1, PB2, PA, or NS, or a functional or immunogenicfragment thereof.
 5. The virus according to claim 4, wherein said one ormore polynucleotides comprise a nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or
 77. 6. The virusaccording to claim 4, wherein said HA protein has an amino acid sequenceshown in SEQ ID NO: 16, 32, 33, 34, 62, or
 78. 7-13. (canceled)
 14. Thevirus according to claim 4, wherein said virus is provided in apharmaceutically acceptable carrier or diluent.
 15. The reassortantvirus according to claim 2, wherein said virus comprises apolynucleotide encoding a hemagglutinin (HA) comprising the amino acidsequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQID NO:62, or SEQ ID NO:78, or an amino acid sequence that comprisesgreater than 95% amino acid sequence identity to SEQ ID NO:16, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78,wherein said HA protein comprises a serine at position 82, a leucine atposition 221, a threonine at position 327, and a threonine at position482 of the amino acid sequence; and/or said virus comprises ahemagglutinin (HA) comprising the amino acid sequence of SEQ ID NO:16,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78,or an amino acid sequence that comprises greater than 95% amino acidsequence identity to SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:62, or SEQ ID NO:78, wherein said HA protein comprisesa serine at position 82, a leucine at position 221, a threonine atposition 327, and a threonine at position 482 of the amino acidsequence.
 16. The reassortant virus according to claim 15, wherein saidpolynucleotide encodes a hemagglutinin (HA) that comprises the aminoacid sequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:62, or SEQ ID NO:78, or an amino acid sequence that comprisesgreater than 95% amino acid sequence identity to SEQ ID NO:16, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78. 17.The reassortant virus according to claim 15, wherein said polynucleotidecomprises the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:31, SEQ IDNO:61, or SEQ ID NO:77, or a nucleotide sequence that comprises 98% orgreater sequence identity to SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:61,or SEQ ID NO:77.
 18. The virus according to claim 4, wherein said viruscomprises a polynucleotide encoding a hemagglutinin (HA) comprising theamino acid sequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:62, or SEQ ID NO:78, or an amino acid sequence thatcomprises greater than 95% amino acid sequence identity to SEQ ID NO:16,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78,wherein said HA protein comprises a serine at position 82, a leucine atposition 221, a threonine at position 327, and a threonine at position482 of the amino acid sequence; and/or said virus comprises ahemagglutinin (HA) comprising the amino acid sequence of SEQ ID NO:16,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78,or an amino acid sequence that comprises greater than 95% amino acidsequence identity to SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:62, or SEQ ID NO:78, wherein said HA protein comprisesa serine at position 82, a leucine at position 221, a threonine atposition 327, and a threonine at position 482 of the amino acidsequence.
 19. The virus according to claim 18, wherein saidpolynucleotide encodes a hemagglutinin (HA) that comprises the aminoacid sequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:62, or SEQ ID NO:78, or an amino acid sequence that comprisesgreater than 95% amino acid sequence identity to SEQ ID NO:16, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78. 20.The virus according to claim 18, wherein said polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:61, orSEQ ID NO:77, or a nucleotide sequence that comprises 98% or greatersequence identity to SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:61, or SEQ IDNO:77.
 21. The reassortant virus according to claim 2, wherein saidreassortant virus is inactivated or attenuated.
 22. The virus accordingto claim 4, wherein said virus is inactivated or attenuated.
 23. Arecombinant viral vector or a polynucleotide vector comprising apolynucleotide that comprises a nucleotide sequence of one or more genesfrom an influenza virus that can infect a canid animal.
 24. The vectoraccording claim 23, wherein said vector comprises a polynucleotide whichencodes a polypeptide comprising the amino acid sequence shown in any ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or78, or a functional and/or immunogenic fragment thereof, or saidpolynucleotide encodes a polypeptide that comprises 95% or greatersequence identity with the amino acid sequence shown in any of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or
 78. 25.The vector according to claim 24, wherein said polynucleotide comprisesthe nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, or 71, or said polynucleotide comprises 95% or greatersequence identity with the nucleotide sequence shown in any of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, or
 71. 26. The vector accordingto claim 23, wherein said vector comprises a polynucleotide encoding ahemagglutinin (HA) comprising the amino acid sequence of SEQ ID NO:16,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78,or an amino acid sequence that comprises greater than 95% amino acidsequence identity to SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:62, or SEQ ID NO:78, wherein said HA protein comprisesa serine at position 82, a leucine at position 221, a threonine atposition 327, and a threonine at position 482 of the amino acidsequence.
 27. The vector according to claim 26, wherein saidpolynucleotide encodes a hemagglutinin (HA) that comprises the aminoacid sequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:62, or SEQ ID NO:78, or an amino acid sequence that comprisesgreater than 95% amino acid sequence identity to SEQ ID NO:16, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:62, or SEQ ID NO:78. 28.The vector according to claim 26, wherein said polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:61, orSEQ ID NO:77, or a nucleotide sequence that comprises 98% or greatersequence identity to SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:61, or SEQ IDNO:77.
 29. The vector according to claim 23, wherein said viral vectoris from adenovirus, avipox, herpesvirus, vaccinia virus, canarypox,entomopox, swinepox, or West Nile virus.
 30. The vector according toclaim 23, wherein said viral vector is a canarypox viral vector.
 31. Thevector according to claim 23, wherein said polynucleotide vector is aviral vector.
 32. A vaccine comprising: a recombinant viral vector or apolynucleotide vector comprising a polynucleotide that comprises anucleotide sequence of one or more genes from an influenza virus thatcan infect a canid animal; a reassortant virus comprising at least onepolypeptide, gene, genomic segment, or a polynucleotide of an influenzavirus that can infect a canid animal; or a virus that comprises one ormore heterologous polypeptides or polynucleotides, wherein said one ormore polypeptides comprises a canine influenza virus protein selectedfrom HA, NA, NP, MA, PB 1, PB2, PA, or NS, or a functional orimmunogenic fragment thereof, or wherein said one or morepolynucleotides encode a canine influenza virus protein selected fromHA, NA, NP, MA, PB 1, PB2, PA, or NS, or a functional or immunogenicfragment thereof.
 33. The vaccine according to claim 32, wherein saidvaccine comprises a pharmaceutically acceptable carrier or diluent. 34.The vaccine according to claim 32, wherein said vaccine comprises anadjuvant.
 35. The vaccine according to claim 32, wherein said virus isinactivated or attenuated.